Nucleic acids encoding neutralizing anti-CCL20 antibodies

ABSTRACT

The present invention relates to novel humanized, chimeric and murine antibodies that have binding specificity for the human CC chemokine ligand 20 (CCL20). The present invention further relates to heavy chains and light chains of said antibodies. The invention also relates to isolated nucleic acids, recombinant vectors and host cells that comprise a sequence which encodes a heavy chain and/or a light chain of said antibodies, and to a method of preparing said antibodies. The anti-CCL20 antibodies of the invention can be used in therapeutic applications to treat, for example, inflammatory and autoimmune disorders and cancer.

This application is a divisional application of U.S. patent applicationSer. No. 13/300,352, filed Nov. 18, 2011, which claims priority fromU.S. Provisional Patent Application No. 61/415,614, filed Nov. 19, 2010.The disclosures of all the aforementioned priority applications areincorporated by reference in their entirety herein.

The instant application contains a Sequence Listing which has beensubmitted in ASCII format via EFS-Web and is hereby incorporated byreference in its entirety. Said ASCII copy, created on Jun. 21, 2013, isnamed “1068060008102.TXT” and is 113,761 bytes in size.

FIELD OF THE INVENTION

The present invention relates to novel humanized, chimeric and murineantibodies specifically directed against the human CC chemokine ligand20 (CCL20). The humanized antibodies of the invention are particularlywell-suited as therapeutics for the treatment of inflammatory andautoimmune diseases.

BACKGROUND OF THE INVENTION

The immune system is a highly sophisticated bio-circuit used by the bodyto discriminate non-self (e.g., foreign organisms or substances) fromself. The detection of non-self in the body can result in inflammation,in which various cellular and molecular components are orchestrated torespond to potentially harmful events caused by the non-self organism orsubstance. Although the inflammatory process helps to protect the bodyfrom foreign attack, de-regulation of the immune system can lead tonegative consequences such as self attack, e.g., autoimmune disease. Byaltering the function of inflammatory molecules such as chemokines, itmay be possible to reduce the initiation and progression of disordersrelating to immune/inflammatory responses.

Chemokines are a family of small (8-10 kDa) proteins that play a pivotalrole in inflammation. During the inflammatory process, chemokines areproduced locally at the site of the noxious stimulus and work as centralplayers to recruit immune cells that express their cognate receptors,seven trans-membrane G protein-coupled receptors (GPCRs). CCL20,alternatively named liver and activation-regulated chemokine (LARC),macrophage inflammatory protein-3 alpha (MIP-3α), or Exodus-1, is asoluble chemokine that is expressed by epithelial cells. Epithelialkeratinocytes and synovium-lining cells are known to produce largeamounts of CCL20 during homeostatic as well as inflammatory andpathological conditions such as cancer, psoriasis, and rheumatoidarthritis. The cognate receptor for CCL20 is CC chemokine receptor 6(CCR6); CCL20 is the only chemokine known to interact with CCR6. Inresponse to the CCL20 signal, immune cells possessing CCR6, such asimmature dendritic cells (DC), effector/memory T-cells, and B-cells,migrate and infiltrate the surrounding tissues, thus activating theinflammatory cascade.

Because CCL20 expression is significantly enhanced in inflammationinduced by inflammatory cytokines such as interleukin 1β (IL-1β) andtumor necrosis factor α (TNF-α), the CCL20-CCR6 interaction is thoughtto play a role in pathological inflammatory processes.

Rheumatoid Arthritis (RA) is one of the most common autoimmune diseases.The first sign of RA is often synovitis, which manifests as a swollen,painful joint. Although the specific factors that initiate synovitisremain unknown, synovium lining epithelial cells and synovialfibroblasts are thought to be primary inducers of the inflammatoryreaction. Synovial fluid from RA patients effectively chemo-attractshuman monocytes and pro-inflammatory T helper 17 (Th17) cells, whichthen induce and exacerbate the RA inflammatory process. Because reactivesynovial cells are capable of producing large amounts of CCL20(particularly under the influence of IL-1β and TNF-α), while CCR6 is themajor receptor of Th17 cells, the CCL20-CCR6 interaction is thought toplay a key role in the inflammatory process.

The CCL20-CCR6 interaction may also play an important role in certaintypes of dermatitis. Psoriasis, for example, initiates with a noxiouspsoriatic event in the skin (induced by environmental and/or geneticfactors) followed by infiltration of Th17 cells. Because CCR6 isexpressed on the surface of Th17 cells, B cells, dendritic cells, andtissue damaging effector T cells, CCL20 may represent the mainchemoattractant for these cell types in psoriasis. Further evidence forthe importance of the CCL20-CCR6 interaction can be found in studiesusing an interleukin 23 (IL-23)-induced mouse model of psoriasis(Hedrick et al., J. Clin. Invest. 119:2317-2329 (2009)). In this model,injection of IL-23 causes interleukin 22 (IL-22)-dependent psoriaticinflammation. However, Ccr6^(−/−) mice did not exhibit psoriasis-likesymptoms when injected with IL-23, indicating that CCR6 is required forthe development of psoriasis.

Human keratinocytes can produce large amounts of CCL20, especially underthe influence of the Th17-derived cytokines interleukin 17 (IL-17),IL-22, and TNF-α. While CCL20 and CCR6 are rarely detected in normalskin, both exhibit increased expression levels in atopic dermatitis andpustular psoriasis. Strong induction of CCL20 and accumulation of CCR6+cells can be observed in microscopic immunohistochemical analysis ofhuman dermatitis lesions. These observations provide additional evidencefor the role of CCL20 and CCR6 in the dermatitis inflammatory process.

Currently available MAb biologics for treating immune disorders can beroughly classified into three groups: inhibitors of immunostimulatorycytokines (e.g., anti-TNF-α MAbs), immune cell eliminators (e.g.,anti-CD20 MAbs), and blockers of accessory molecules (e.g. Abatacept).These biologics may be useful in the treatment of inflammatory diseases;however, due to primary non-responsiveness or a gradual decline inresponse rate to these treatments, there is an urgent need foralternative biologics with novel mechanisms of action to meet themedical needs of patients with, e.g., CCL20/CCR6-mediated disorders. Theantibodies of the subject invention represent such alternativebiologics.

SUMMARY OF THE INVENTION

The present invention relates to neutralizing anti-CCL20 antibodies, orantigen-binding portions thereof. In certain embodiments, the anti-CCL20antibody is a humanized anti-human CCL20 antibody, which may comprisethe complementarity determining regions (CDRs) of mouse anti-human CCL20antibodies. In some embodiments, the anti-CCL20 antibody is a mouse orchimeric anti-human CCL20 antibody or an antigen-binding portionthereof.

In some embodiments, the anti-CCL20 antibody or antigen-binding portionthereof binds to human CCL20. In some embodiments, the antibody orportion does not bind to human CCL16.

The antibodies of the invention specifically bind CCL20. In someembodiments, the anti-CCL20 antibody or antigen-binding portion thereofbinds to cynomolgus and/or rhesus CCL20 as well as human CCL20. In someembodiments, the anti-CCL20 antibody or antigen-binding portion thereofdoes not bind to mouse and/or rat CCL20. In one embodiment, theanti-CCL20 antibody or antigen-binding portion binds to human,cynomolgus, and rhesus CCL20, but not to mouse and rat CCL20.

In some embodiments, the anti-CCL20 antibody or antigen-binding portionthereof has a binding affinity (K_(D)) for human CCL20 of less than:

-   -   1 nM, 500 pM, 100 pM, 90 pM, 80 M, 70 pM, 60 pM, or 50 pM using        a monovalent surface plasmon resonance assay, or    -   1 nM, 500 pM, 100 pM, 75 pM, 50 pM, 25 pM, 20 pM, 15 pM, 14 pM,        13 pM, 12 pM, 11 pM, 10 pM, 9 pM, 8 pM, 7 pM, 6 pM, or 5 pM        using a bivalent surface plasmon resonance assay.        In certain embodiments, the antibody or portion has a binding        affinity for human CCL20 greater than that of human CCR6.

In some embodiments, the anti-CCL20 antibody or antigen-binding portionthereof has a k_(a) for human CCL20 of less than 100, 90, 80, 70, 60,50, 40, or 30 (×10⁵ sec⁻¹), as determined by bivalent surface plasmonresonance. In some embodiments, the anti-CCL20 antibody orantigen-binding portion thereof has a k_(d) for human CCL20 of less than10, 9, 8, 7, 6, 5, 4, or 3 (×10⁻⁵ sec⁻¹), as determined by bivalentsurface plasmon resonance.

In some embodiments, the anti-CCL20 antibody or antigen-binding portionthereof has a k_(a) for rhesus CCL20 of less than 100, 90, 80, 70, 60,50, 40, or 30 (×10⁵ M⁻¹ sec⁻¹), as determined by bivalent surfaceplasmon resonance. In some embodiments, the anti-CCL20 antibody orantigen-binding portion thereof has a k_(d) for rhesus CCL20 of lessthan 50, 40, 30, 20, 10, 9, 8, 7, 6, 5, 4, 3, or 2 (×10⁻⁵ sec⁻¹), asdetermined by bivalent surface plasmon resonance. In some embodiments,the anti-CCL20 antibody or antigen-binding portion thereof has a K_(D)for rhesus CCL20 of less than 100, 90, 80, 70, 60, 50, 40, 30, 20, or 10μM, as determined by bivalent surface plasmon resonance.

In some embodiments, the anti-CCL20 antibody or antigen-binding portionthereof has a k_(a) for cynomolgus CCL20 of less than 500, 400, 300,200, 100, 90, 80, 70, 60, 50, 40, or 30 (×10⁵ M⁻¹ sec⁻¹), as determinedby bivalent surface plasmon resonance. In some embodiments, theanti-CCL20 antibody or antigen-binding portion thereof has a k_(d) forcynomolgus CCL20 of less than 50, 40, 30, 20, 19, 18, 17, 16, 15, 14,13, 12, 11, 10, 9, 8, 7, or 6 (×10⁻⁵ sec⁻¹), as determined by bivalentsurface plasmon resonance. In some embodiments, the anti-CCL20 antibodyor antigen-binding portion thereof has a K_(D) for cynomolgus CCL20 ofless than 100, 90, 80, 70, 60, 50, 40, 30, 20, 19, 18, 17, 16, or 15 pM,as determined by bivalent surface plasmon resonance.

In some embodiments, the anti-CCL20 antibody or antigen-binding portionthereof binds to human CCL20 with an EC₅₀ of less than 100, 90, 80, 70,60, 50, 40, 39, 38, 37, 36, 35, or 34 pM. In some embodiments, theanti-CCL20 antibody or antigen-binding portion thereof binds to rhesusCCL20 with an EC₅₀ of less than 500, 400, 300, 200, 150, 140, 130, 120,110, 100, 90, 80, 70, 65, 60, 59, 58, 57, 56, 55, 54, 53, 52, 51, or 50pM. In some embodiments, the anti-CCL20 antibody or antigen-bindingportion thereof binds to cynomolgus CCL20 with an EC₅₀ of less than 500,400, 350, 300, 250, 200, 190, 180, 170, 160, 150, 140, 130, 120, 110,109, 108, 107, 106, 105, 104, 103, or 102 pM.

In some embodiments, the anti-CCL20 antibody or antigen-binding portionthereof has a selectivity for human CCL20 over other human chemokines,including but not limited to CX3CL1, CXCL1, CXCL2, CXCL4, CXCL8, CXCL9,CXCL10, CXCL12, CXCL13, CXCL16, CCL1, CCL2, CCL3, CCL4, CCL5, CCL7,CCL11, CCL13, CCL16, CCL17, CCL19, CCL21, CCL22, CCL24, CCL25, CCL27,CCL28, and/or XCL1. In certain embodiments, the anti-CCL20 antibody orantigen-binding portion has a selectivity for human CCL20 over all ofsaid chemokines.

In some embodiments, the anti-CCL20 antibody or antigen-binding portionthereof reduces human CCL20-induced chemotaxis of human or mouse CCR6+cells with an IC₅₀ of less than 20, 19, 18, 17, 16, 15, 14, 13, 12, 11,10, 9, 8, 7, 6, 5, 4, 3, 2, 1.7, 1.6, 1.5, 1.4, 1.3, 1.2, or 1.1 nM. Incertain embodiments, the anti-CCL20 antibody or antigen-binding portionthereof reduces human CCL20-induced chemotaxis of human or mouse CCR6+cells with an IC₅₀ of less than 1.7, 1.6, 1.5, 1.4, 1.3, 1.2, or 1.1 nM.In some embodiments, the anti-CCL20 antibody or antigen-binding portionthereof reduces human CCL20-induced chemotaxis of human or mouse CCR6+cells with an IC₉₀ of less than 45, 40, 35, 30, 25, 20, 19, 18, 17, 16,15, 14, 13, 12, 11, 10, 9, 8, 7, 6, 5.5, 5.4, 5.3, 5.2, 5.1, 4.9, 4.8,4.7, or 4.6 nM. In certain embodiments, the anti-CCL20 antibody orantigen-binding portion thereof reduces human CCL20-induced chemotaxisof human or mouse CCR6+ cells with an IC₉₀ of less than 6, 5.5, 5.4,5.3, 5.2, 5.1, 4.9, 4.8, 4.7, or 4.6 nM. In some embodiments, theanti-CCL20 antibody or antigen-binding portion thereof reduces humanCCL20-induced chemotaxis of human or mouse CCR6+ cells with an IC₉₅ ofless than 70, 65, 60, 55, 50, 45, 40, 35, 30, 25, 20, 15, 10, 9, 8, or 7nM. In certain embodiments, the anti-CCL20 antibody or antigen-bindingportion thereof reduces human CCL20-induced chemotaxis of human or mouseCCR6+ cells with an IC₉₅ of less than 10, 9.9, 9.8, 9.7, 9.6, 9.5, 9.4,9.3, 9.2, 9.1, or 9 nM.

In some embodiments, the anti-CCL20 antibody or antigen-binding portionthereof reduces human CCL20-induced chemotaxis of human or mouse CCR6+cells in vitro.

In some embodiments, the anti-CCL20 antibody or antigen-binding portionthereof reduces human CCL20-induced chemotaxis of human or mouse CCR6+cells in vivo.

In some embodiments, the anti-CCL20 antibody or antigen-binding portionthereof reduces the progression of arthritis symptoms (such as articularlesions of the extremities distal to the elbow or knee and degree oferythema and swelling) in a subject, e.g., in a collagen-inducedarthritis (CIA) and/or a glucose-6-phosphate isomerase (G6PI)-inducedarthritis mouse model. In some embodiments, the antibody orantigen-binding portion thereof reduces bone lesions in a subject,including osteoporosis, bone erosion, and/or new bone formation, e.g.,in a CIA model. In some embodiments, the antibody or antigen-bindingportion reduces cartilage oligomeric matrix protein (COMP) serum levelsin a subject, e.g., in a CIA model. In some embodiments, the antibody orantigen-binding portion thereof reduces mRNA levels of receptoractivator for nuclear factor κB ligand (RANKL), receptor activator fornuclear factor κB (RANK), tartrate resistant acid phosphatase (TRAP),and/or cathepsin K in a subject, e.g., in mouse paws in a CIA model. Insome embodiments, the antibody or antigen-binding portion thereofreduces atopic dermatitis symptoms (e.g., dryness, scale, erythema,oozing/crusting, and/or excoriation) in a subject, for example, symptomsof oxazolone-induced atopic dermatitis in NC/Nga strain mice. In someembodiments, the antibody or antigen-binding portion reduces allergiccontact dermatitis symptoms in a subject, for example, symptoms ofdinitrofluorobenzene (DNFB)-induced allergic contact dermatitis in amouse model.

In some embodiments, the anti-CCL20 antibody or antigen-binding portionthereof comprises a heavy chain whose CDR3 (H-CDR3) comprises thesequence of SEQ ID NO: 67 or 68.

In some embodiments, the anti-CCL20 antibody or antigen-binding portionthereof comprises a light chain whose CDR3 (L-CDR3) comprises thesequence of SEQ ID NO 75.

In certain embodiments, the anti-CCL20 antibody or antigen-bindingportion thereof comprises an H-CDR3 whose sequence comprises SEQ ID NO:67 and an L-CDR3 whose sequence comprises SEQ ID NO: 75; or an H-CDR3whose sequence comprises SEQ ID NO: 68 and an L-CDR3 whose sequencecomprises SEQ ID NO: 75.

In some embodiments, the anti-CCL20 antibody or antigen-binding portionthereof comprises a heavy chain whose CDR1 (H-CDR1), CDR2 (H-CDR2), andCDR3 (H-CDR3) respectively comprise the sequences of:

-   -   SEQ ID NOS: 60, 63, and 67;    -   SEQ ID NOS: 60, 64, and 67;    -   SEQ ID NOS: 61, 65, and 68;    -   SEQ ID NOS: 77, 79, and 67; or    -   SEQ ID NOS: 78, 80, and 68.

In some embodiments, the anti-CCL20 antibody or antigen-binding portionthereof comprises a light chain whose CDR1 (L-CDR1), CDR2 (L-CDR2), andCDR3 (L-CDR3) respectively comprise the sequences of:

-   -   SEQ ID NOS: 70, 73, and 75; or    -   SEQ ID NOS: 71, 73, and 75.

In some embodiments, the anti-CCL20 antibody or antigen-binding portionthereof comprises:

-   -   an H-CDR1 comprising a sequence selected from the group        consisting of SEQ ID NOS: 60-62, 77, and 78;    -   an H-CDR2 comprising a sequence selected from the group        consisting of SEQ ID NOS: 63-66 and 79-81;    -   an H-CDR3 comprising the sequence of SEQ ID NO: 67 or 68;    -   an L-CDR1 comprising a sequence selected from the group        consisting of SEQ ID NOS: 70-72;    -   an L-CDR2 comprising the sequence of SEQ ID NO: 73 or 74; or    -   an L-CDR3 comprising SEQ ID NO: 75;        or any combination thereof.

In certain embodiments, the anti-CCL20 antibody or antigen-bindingportion thereof comprises an H-CDR1, H-CDR2, H-CDR3, L-CDR1, L-CDR2, andL-CDR3 respectively comprising the sequences of:

-   -   SEQ ID NOS: 60, 63, 67, 70, 73, and 75;    -   SEQ ID NOS: 60, 64, 67, 70, 73, and 75;    -   SEQ ID NOS: 61, 65, 68, 70, 73, and 75;    -   SEQ ID NOS: 77, 79, 67, 70, 73, and 75;    -   SEQ ID NOS: 78, 80, 68, 70, 73, and 75;    -   SEQ ID NOS: 60, 63, 67, 71, 73, and 75;    -   SEQ ID NOS: 60, 64, 67, 71, 73, and 75;    -   SEQ ID NOS: 61, 65, 68, 71, 73, and 75;    -   SEQ ID NOS: 77, 79, 67, 71, 73, and 75; or    -   SEQ ID NOS: 78, 80, 68, 71, 73, and 75.

In some embodiments, the anti-CCL20 antibody or antigen-binding portionthereof comprises a heavy chain variable domain comprising a sequenceselected from the group consisting of SEQ ID NOS: 9-14. In someembodiments, the anti-CCL20 antibody or antigen-binding portion thereofcomprises a heavy chain variable domain comprising a sequence selectedfrom the group consisting of SEQ ID NOS: 39, 41, and 43.

In some embodiments, the anti-CCL20 antibody or antigen-binding portionthereof comprises a light chain variable domain comprising the sequenceof SEQ ID NO: 15 or 16. In some embodiments, the anti-CCL20 antibody orantigen-binding portion thereof comprises a light chain variable domaincomprising a sequence selected from the group consisting of SEQ ID NOS:40, 42, and 44.

In certain embodiments, the anti-CCL20 antibody or antigen-bindingportion thereof comprises a heavy chain variable domain and a lightchain variable domain respectively comprising the sequences of:

-   -   SEQ ID NO: 9 and SEQ ID NO: 15;    -   SEQ ID NO: 10 and SEQ ID NO: 15;    -   SEQ ID NO: 11 and SEQ ID NO: 15;    -   SEQ ID NO: 12 and SEQ ID NO: 15;    -   SEQ ID NO: 13 and SEQ ID NO: 15;    -   SEQ ID NO: 14 and SEQ ID NO: 15;    -   SEQ ID NO: 9 and SEQ ID NO: 16;    -   SEQ ID NO: 10 and SEQ ID NO: 16;    -   SEQ ID NO: 11 and SEQ ID NO: 16;    -   SEQ ID NO: 12 and SEQ ID NO: 16;    -   SEQ ID NO: 13 and SEQ ID NO: 16; or    -   SEQ ID NO: 14 and SEQ ID NO: 16.

In certain embodiments, the anti-CCL20 antibody or antigen-bindingportion thereof comprises a heavy chain variable domain and a lightchain variable domain respectively comprising the sequences of:

-   -   SEQ ID NO: 39 and SEQ ID NO: 40;    -   SEQ ID NO: 39 and SEQ ID NO: 42;    -   SEQ ID NO: 39 and SEQ ID NO: 44;    -   SEQ ID NO: 41 and SEQ ID NO: 40;    -   SEQ ID NO: 41 and SEQ ID NO: 42;    -   SEQ ID NO: 41 and SEQ ID NO: 44;    -   SEQ ID NO: 43 and SEQ ID NO: 40;    -   SEQ ID NO: 43 and SEQ ID NO: 42; or    -   SEQ ID NO: 43 and SEQ ID NO: 44.

In some embodiments, the anti-CCL20 antibody or antigen-binding portionthereof comprises a heavy chain having a sequence selected from thegroup consisting of SEQ ID NOS: 1-6 and 108 without the signal sequence(if present), and optionally without the C-terminal lysine. In someembodiments, the anti-CCL20 antibody or antigen-binding portion thereofcomprises a light chain having a sequence selected from the groupconsisting of SEQ ID NOS: 7, 8, 110, and 112 without the signal sequence(if present). In certain embodiments, the anti-CCL20 antibody comprisesa heavy chain and a light chain respectively having the sequences of:

-   -   SEQ ID NO: 1 and SEQ ID NO: 7;    -   SEQ ID NO: 2 and SEQ ID NO: 7;    -   SEQ ID NO: 3 and SEQ ID NO: 7;    -   SEQ ID NO: 4 and SEQ ID NO: 7;    -   SEQ ID NO: 5 and SEQ ID NO: 7;    -   SEQ ID NO: 6 and SEQ ID NO: 7;    -   SEQ ID NO: 1 and SEQ ID NO: 8;    -   SEQ ID NO: 2 and SEQ ID NO: 8;    -   SEQ ID NO: 3 and SEQ ID NO: 8;    -   SEQ ID NO: 4 and SEQ ID NO: 8;    -   SEQ ID NO: 5 and SEQ ID NO: 8;    -   SEQ ID NO: 6 and SEQ ID NO: 8;    -   SEQ ID NO: 108 and SEQ ID NO: 110; or    -   SEQ ID NO: 108 and 112;        both of said sequences without the signal sequence, SEQ ID NOS:        1-6 and 108 optionally without the C-terminal lysine.

The present invention also provides an anti-CCL20 antibody or anantigen-binding portion thereof that comprises a heavy chain variabledomain encoded by a sequence selected from the group consisting of SEQID NOS: 25-30. In some embodiments, the anti-CCL20 antibody orantigen-binding portion thereof comprises a heavy chain variable domainencoded by a sequence selected from the group consisting of SEQ ID NOS:51, 53, and 55.

Further, the present invention provides an anti-CCL20 antibody or anantigen-binding portion thereof that comprises a light chain variabledomain encoded by the sequence of SEQ ID NO: 31 or 32. In someembodiments, the anti-CCL20 antibody or antigen-binding portion thereofcomprises a light chain variable domain encoded by a sequence selectedfrom the group consisting of SEQ ID NOS: 52, 54, and 56.

In certain embodiments, the anti-CCL20 antibody or antigen-bindingportion thereof comprises a heavy chain variable domain and a lightchain variable domain respectively encoded by the sequences of:

-   -   SEQ ID NO: 25 and SEQ ID NO: 31;    -   SEQ ID NO: 26 and SEQ ID NO: 31;    -   SEQ ID NO: 27 and SEQ ID NO: 31;    -   SEQ ID NO: 28 and SEQ ID NO: 31;    -   SEQ ID NO: 29 and SEQ ID NO: 31;    -   SEQ ID NO: 30 and SEQ ID NO: 31;    -   SEQ ID NO: 25 and SEQ ID NO: 32;    -   SEQ ID NO: 26 and SEQ ID NO: 32;    -   SEQ ID NO: 27 and SEQ ID NO: 32;    -   SEQ ID NO: 28 and SEQ ID NO: 32;    -   SEQ ID NO: 29 and SEQ ID NO: 32; or    -   SEQ ID NO: 30 and SEQ ID NO: 32.

In certain embodiments, the anti-CCL20 antibody or antigen-bindingportion thereof comprises a heavy chain variable domain and a lightchain variable domain respectively encoded by the sequences of:

-   -   SEQ ID NO: 51 and SEQ ID NO: 52;    -   SEQ ID NO: 51 and SEQ ID NO: 54;    -   SEQ ID NO: 51 and SEQ ID NO: 56;    -   SEQ ID NO: 53 and SEQ ID NO: 52;    -   SEQ ID NO: 53 and SEQ ID NO: 54;    -   SEQ ID NO: 53 and SEQ ID NO: 56;    -   SEQ ID NO: 55 and SEQ ID NO: 52;    -   SEQ ID NO: 55 and SEQ ID NO: 54; or    -   SEQ ID NO: 55 and SEQ ID NO: 56.

In some embodiments, the anti-CCL20 antibody or antigen-binding portionthereof comprises a heavy chain encoded by a sequence selected from thegroup consisting of SEQ ID NOS: 17-22 and 109, said sequence lacking thesequence encoding a signal sequence (if present) and optionally lackingthe sequence encoding the C-terminal lysine. In some embodiments, theanti-CCL20 antibody or antigen-binding portion thereof comprises a lightchain encoded by a sequence selected from the group consisting of SEQ IDNOS: 23, 24, 111, and 113, said sequence lacking the sequence encoding asignal sequence (if present). In certain embodiments, the anti-CCL20antibody comprises a heavy chain and a light chain respectively encodedby the sequences of:

-   -   SEQ ID NO: 17 and SEQ ID NO: 23;    -   SEQ ID NO: 18 and SEQ ID NO: 23;    -   SEQ ID NO: 19 and SEQ ID NO: 23;    -   SEQ ID NO: 20 and SEQ ID NO: 23;    -   SEQ ID NO: 21 and SEQ ID NO: 23;    -   SEQ ID NO: 22 and SEQ ID NO: 23;    -   SEQ ID NO: 17 and SEQ ID NO: 24;    -   SEQ ID NO: 18 and SEQ ID NO: 24;    -   SEQ ID NO: 19 and SEQ ID NO: 24;    -   SEQ ID NO: 20 and SEQ ID NO: 24;    -   SEQ ID NO: 21 and SEQ ID NO: 24;    -   SEQ ID NO: 22 and SEQ ID NO: 24;    -   SEQ ID NO: 109 and SEQ ID NO: 111; or    -   SEQ ID NO: 109 and SEQ ID NO: 113;        both of said sequences lacking the sequence encoding a signal        sequence (if present), SEQ ID NOS: 17-22 and 109 optionally        lacking the sequence encoding the C-terminal lysine.

In some embodiments, the anti-CCL20 antibody or antigen-binding portionthereof is humanized or chimeric. In some embodiments, the frameworkregions of the heavy chain of the humanized anti-CCL20 antibody orportion utilize an IGHV1-46*03 human germline gene (See, e.g., Altschulet al., “Gapped BLAST and PSI-BLAST: a new generation of proteindatabase search programs”, Nucleic Acids Res. 25:3389-3402 (1997)). Incertain embodiments, the heavy chain framework regions have at least 50%homology to the framework regions of the IGHV1-46*03 human germlinegene. For example, the heavy chain framework regions may be at least50%, at least 60%, at least 70%, at least 80%, at least 90%, at least95%, at least 98%, at least 99%, or even 100% identical to the frameworkregions of the IGHV1-46*03 human germline gene. In some embodiments, theframework regions of the light chain of the humanized anti-CCL20antibody or portion utilize an IGKV1D-39*01 human germline gene (seeAltschul et al., supra). In certain embodiments, the light chainframework regions have at least 50% homology to the framework regions ofthe IGKV1D-39*01 human germline gene. For example, the light chainframework regions may be at least 50%, at least 60%, at least 70%, atleast 80%, at least 90%, at least 95%, at least 98%, at least 99%, oreven 100% identical to the framework regions of the IGKV1D-39*01 humangermline gene.

In some embodiments, the anti-CCL20 antibody or antigen-binding portionthereof (to the extent that said portion comprises at least part of aheavy chain constant region) is an IgG, an IgM, an IgE, an IgA, or anIgD molecule. In certain embodiments, the anti-CCL20 antibody orantigen-binding portion thereof is of the IgG1, IgG2, IgG3, or IgG4subtype.

In some embodiments, the anti-CCL20 antibody is a monoclonal antibody.

The present invention also provides an anti-CCL20 antibody or anantigen-binding portion thereof that binds to an epitope of human CCL20located in the N-loop and/or 132-133 hairpin region of the molecule. Insome embodiments, said anti-CCL20 antibody or portion binds to the sameepitope on human CCL20 as an antibody or portion as described herein.Further, the present invention provides an anti-CCL20 antibody or anantigen-binding portion thereof that competes or cross-competes forbinding to human CCL20 with an antibody or portion described herein.

In some embodiments, the human CCL20 epitope bound by the antibody orantigen-binding portion thereof of the invention comprises one or moreamino acid sequences selected from the group consisting of:

-   -   residues 7-9 of SEQ ID NO: 84;    -   residues 10-19 of SEQ ID NO: 84;    -   residues 20-21 of SEQ ID NO: 84; and    -   residues 20-22 of SEQ ID NO: 84,        or the residues of wild-type human CCL20 corresponding to said        residues of SEQ ID NO: 84.

In some embodiments, said human CCL20 epitope further comprises one ormore amino acid sequences selected from the group consisting of:

-   -   residues 39-55 of SEQ ID NO: 84;    -   residues 39-57 of SEQ ID NO: 84;    -   residues 56-67 of SEQ ID NO: 84; and    -   residues 61-70 of SEQ ID NO: 84,        or the residues of wild-type human CCL20 corresponding to said        residues of SEQ ID NO: 84.

In certain embodiments, said human CCL20 epitope comprises anycombination of the amino acid sequences consisting of residues 7-9,10-19, 20-22, 39-55, 56-57, and 61-70 of SEQ ID NO: 84, or the residuesof wild-type human CCL20 corresponding to said residues of SEQ ID NO:84.

In certain embodiments, said human CCL20 epitope comprises residues 7-9,10-19, and 20-22 of SEQ ID NO: 84, or the residues of wild-type humanCCL20 corresponding to said residues of SEQ ID NO: 84. In certainembodiments, said human CCL20 epitope comprises residues 7-9, 10-19,20-22, 39-55, 56-57, and 61-70 of SEQ ID NO: 84, or the residues ofwild-type human CCL20 corresponding to said residues of SEQ ID NO: 84.In certain embodiments, said human CCL20 epitope comprises residues 7-9,10-19, 20-22, 39-57, and 61-70 of SEQ ID NO: 84, or the residues ofwild-type human CCL20 corresponding to said residues of SEQ ID NO: 84.

In certain embodiments, said human CCL20 epitope comprises residues 7-9,10-19, and 20-21 of SEQ ID NO: 84, or the residues of wild-type humanCCL20 corresponding to said residues of SEQ ID NO: 84. In certainembodiments, said human CCL20 epitope comprises residues 7-9, 10-19,20-21, 39-55, 56-67, and 61-70 of SEQ ID NO: 84, or the residues ofwild-type human CCL20 corresponding to said residues of SEQ ID NO: 84.In certain embodiments, said human CCL20 epitope comprises residues 7-9,10-19, 20-21, 39-57, and 61-70 of SEQ ID NO: 84, or the residues ofwild-type human CCL20 corresponding to said residues of SEQ ID NO: 84.

In some embodiments, the human CCL20 epitope bound by the antibody orantigen-binding portion thereof comprises one or more amino acidresidues of SEQ ID NO: 84 (or the residues of wild-type human CCL20corresponding to said residues of SEQ ID NO: 84) selected from residues7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 39, 40, 41,42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59,60, 61, 62, 63, 64, 65, 66, 67, 68, 69, and 70, or any combinationthereof.

The present invention also provides an antigen-binding portion of any ofthe anti-CCL20 antibodies described herein. Said antigen-binding portionmay be, e.g., a single chain antibody, Fv, Fab, Fab′, F(ab′)₂, Fd,single chain Fv molecule (scFv), bispecific single chain Fv dimer,polyvalent single chain Fv multimer, diabody, domain-deleted antibody,or single domain antibody (dAb).

The present invention also provides a variant of an antibody orantigen-binding portion as described herein, wherein said variantdiffers from the antibody or portion by 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10amino acid substitutions.

In one aspect, the present invention provides an antibody directedagainst human CCL20, wherein the heavy chain variable domain and thelight chain variable domain of said antibody respectively comprise thesequences of:

-   -   SEQ ID NOS: 9 and 15;    -   SEQ ID NOS: 9 and 16;    -   SEQ ID NOS: 10 and 16;    -   SEQ ID NOS: 11 and 15;    -   SEQ ID NOS: 11 and 16;    -   SEQ ID NOS: 12 and 16;    -   SEQ ID NOS: 13 and 16; or    -   SEQ ID NOS: 14 and 16.

In one aspect, the present invention provides an antibody whose heavychain comprises SEQ ID NO: 1 and whose light chain comprises SEQ ID NO:7, wherein said amino acid sequences lack signal sequences.

In another aspect, the present invention provides an antibody whoseheavy chain comprises SEQ ID NO: 1 without the C-terminal lysine andwhose light chain comprises SEQ ID NO: 7, wherein said amino acidsequences lack signal sequences.

In one aspect, the present invention provides an antibody whose heavychain comprises SEQ ID NO: 1 and whose light chain comprises SEQ ID NO:8, wherein said amino acid sequences lack signal sequences.

In another aspect, the present invention provides an antibody whoseheavy chain comprises SEQ ID NO: 1 without the C-terminal lysine andwhose light chain comprises SEQ ID NO: 8, wherein said amino acidsequences lack signal sequences.

In one aspect, the present invention provides an antibody whose heavychain comprises SEQ ID NO: 2 and whose light chain comprises SEQ ID NO:8, wherein said amino acid sequences lack signal sequences.

In another aspect, the present invention provides an antibody whoseheavy chain comprises SEQ ID NO: 2 without the C-terminal lysine andwhose light chain comprises SEQ ID NO: 8, wherein said amino acidsequences lack signal sequences.

In one aspect, the present invention provides an antibody whose heavychain comprises SEQ ID NO: 3 and whose light chain comprises SEQ ID NO:7, wherein said amino acid sequences lack signal sequences.

In another aspect, the present invention provides an antibody whoseheavy chain comprises SEQ ID NO: 3 without the C-terminal lysine andwhose light chain comprises SEQ ID NO: 7, wherein said amino acidsequences lack signal sequences.

In one aspect, the present invention provides an antibody whose heavychain comprises SEQ ID NO: 3 and whose light chain comprises SEQ ID NO:8, wherein said amino acid sequences lack signal sequences.

In another aspect, the present invention provides an antibody whoseheavy chain comprises SEQ ID NO: 3 without the C-terminal lysine andwhose light chain comprises SEQ ID NO: 8, wherein said amino acidsequences lack signal sequences.

In one aspect, the present invention provides an antibody whose heavychain comprises SEQ ID NO: 4 and whose light chain comprises SEQ ID NO:8, wherein said amino acid sequences lack signal sequences.

In another aspect, the present invention provides an antibody whoseheavy chain comprises SEQ ID NO: 4 without the C-terminal lysine andwhose light chain comprises SEQ ID NO: 8, wherein said amino acidsequences lack signal sequences.

In one aspect, the present invention provides an antibody whose heavychain comprises SEQ ID NO: 5 and whose light chain comprises SEQ ID NO:8, wherein said amino acid sequences lack signal sequences.

In another aspect, the present invention provides an antibody whoseheavy chain comprises SEQ ID NO: 5 without the C-terminal lysine andwhose light chain comprises SEQ ID NO: 8, wherein said amino acidsequences lack signal sequences.

In one aspect, the present invention provides an antibody whose heavychain comprises SEQ ID NO: 6 and whose light chain comprises SEQ ID NO:8, wherein said amino acid sequences lack signal sequences.

In another aspect, the present invention provides an antibody whoseheavy chain comprises SEQ ID NO: 6 without the C-terminal lysine andwhose light chain comprises SEQ ID NO: 8, wherein said amino acidsequences lack signal sequences.

In one aspect, the present invention provides an antibody whose heavychain comprises SEQ ID NO: 108 and whose light chain comprises SEQ IDNO: 110.

In another aspect, the present invention provides an antibody whoseheavy chain comprises SEQ ID NO: 108 without the C-terminal lysine andwhose light chain comprises SEQ ID NO: 110.

In one aspect, the present invention provides an antibody whose heavychain comprises SEQ ID NO: 108 and whose light chain comprises SEQ IDNO: 112.

In another aspect, the present invention provides an antibody whoseheavy chain comprises SEQ ID NO: 108 without the C-terminal lysine andwhose light chain comprises SEQ ID NO: 112.

In another aspect, the present invention provides one or more nucleicacid molecules, e.g., isolated nucleic acid molecules, encoding a heavychain or an antigen-binding portion thereof or a light chain or anantigen-binding portion thereof, wherein said heavy chain and lightchain or antigen-binding portions thereof may associate to form ananti-CCL20 antibody or an antigen-binding portion thereof. In someembodiments, the nucleic acid molecule encoding the heavy chain or anantigen-binding portion thereof comprises a nucleotide sequence selectedfrom the group consisting of SEQ ID NOS: 17-22, 25-30, and 109, or saidnucleotide sequence without the sequence encoding a signal sequence, ifpresent. The nucleotide sequences of SEQ ID NOS: 17-22 and 109 may alsooptionally lack the sequence encoding the C-terminal lysine, if present.In some embodiments, the nucleic acid molecule encoding the light chainor an antigen-binding portion thereof comprises a nucleotide sequenceselected from the group consisting of SEQ ID NOS: 23, 24, 31, 32, 111,and 113, or said nucleotide sequence without the sequence encoding asignal sequence, if present. In some embodiments, the one or morenucleic acid molecules encode the heavy chain or an antigen-bindingportion thereof and the light chain or an antigen-binding portionthereof of an antibody or portion as described herein. In certainembodiments, one nucleic acid molecule encodes both said heavy chain oran antigen-binding portion thereof and said light chain or anantigen-binding portion thereof.

In one embodiment, the nucleic acid molecule encoding the heavy chain oran antigen-binding portion thereof, the nucleic acid molecule encodingthe light chain or an antigen-binding portion thereof, or nucleic acidmolecule(s) encoding the heavy chain and the light chain orantigen-binding portions thereof, are used for treating a subject inneed thereof.

In another aspect, the present invention provides a vector comprisingnucleic acid sequence(s) encoding the heavy chain or an antigen-bindingportion thereof, the light chain or an antigen-binding portion thereof,or both, of an antibody or portion as described herein.

In another aspect, the present invention provides a cell expressing theheavy chain or an antigen-binding portion thereof, the light chain or anantigen-binding portion thereof, or both, of an antibody or portion asdescribed herein.

In another aspect, the present invention provides a method for making anantibody or portion as described herein, comprising maintaining a cellas described herein under conditions appropriate for expression of theantibody or portion. In some embodiments, the method comprises the stepof isolating the antibody or portion.

The present invention also provides a method of producing a hybridomathat secretes an anti-CCL20 antibody. In certain embodiments, thehybridoma produces an antibody that binds to wild-type human CCL20. Theinvention also provides a hybridoma produced by the methods of theinvention. Optionally, the monoclonal antibody secreted by the hybridomais collected and can be further purified (e.g., substantially purified,isolated). In other embodiments, the method further comprisesdetermining the nucleotide sequence of the monoclonal antibody secretedby the hybridoma.

In another aspect, the present invention provides a compositioncomprising an antibody or portion as described herein and apharmaceutically acceptable vehicle or carrier.

In one aspect, the antibody or portion as described herein is used as amedicament. In certain embodiments, the antibody or portion as describedherein is used for treating a subject in need thereof. In particularembodiments, the antibody or portion is used to treat a CCR6-associatedcondition, a CCL20 associated condition or both. Such conditions includebut are not limited to inflammatory and autoimmune diseases,particularly arthritis, especially rheumatoid arthritis, atopic orallergic dermatitis, and psoriasis. In another particular embodiment,the antibody or portion is used to treat cancer. The antibody or portionmay be used to treat, e.g., Grave's disease, vitiligo, hyperthyroidism,rheumatoid arthritis, psoriasis, atopic dermatitis, contact dermatitis,Crohn's disease, inflammatory bowel disease, B-cell malignancies, breastadenocarcinoma, chronic hepatitis, contact dermatitis, glioblastoma,hepatocellular carcinoma, human papillomavirus infection of the cervix,mycosis fungoides, pancreatic adenocarcinoma, periodontal disease,thyroid papillary carcinoma, pustulosis palmaris et plantaris,conditions associated with maculopapular exanthema, epidermolysisbullosa, alopecia greata, multiple sclerosis, polymyositis,dermatomyositis, Behcet's disease, acute generalized exanthematouspustulosis, vasculitides, juvenile idiopathic arthritis, sarcoidosis,bronchial asthma, allergic rhinitis, renal allograft rejection,graft-versus-host disease, liver allograft rejection, chronicobstructive pulmonary disease, cystic fibrosis, glomerulonephritis,respiratory syncytial virus infection, multiple myeloma, and/orLangerhans cell histiocytosis.

In some embodiments, the anti-CCL20 antibody or portion is used toreduce CCL20-mediated chemotaxis of CCR6+ cells in a subject in needthereof.

In some embodiments, the present invention provides:

-   1. A monoclonal anti-human CCL20 antibody or an antigen-binding    portion thereof, wherein said antibody comprises a heavy chain whose    complementarity determining region 3 (CDR3) comprises SEQ ID NO: 67    or 68.-   2. The monoclonal antibody or antigen-binding portion of embodiment    1, wherein the complementarity determining region 1 (CDR1),    complementarity determining region 2 (CDR2), and complementarity    determining region 3 (CDR3) of said heavy chain respectively    comprise amino acid sequences selected from the group consisting of:    -   a) SEQ ID NOS: 60, 64, and 67;    -   b) SEQ ID NOS: 60, 63, and 67;    -   c) SEQ ID NOS: 61, 65, and 68;    -   d) SEQ ID NOS: 77, 79, and 67; and    -   e) SEQ ID NOS: 78, 80, and 68.-   3. A monoclonal anti-human CCL20 antibody or an antigen-binding    portion thereof, wherein said antibody comprises a light chain whose    complementarity determining region 3 (CDR3) comprises SEQ ID NO: 75.-   4. The monoclonal antibody or antigen-binding portion of embodiment    3, wherein the complementarity determining region 1 (CDR1),    complementarity determining region 2 (CDR2), and complementarity    determining region 3 (CDR3) of said light chain respectively    comprise amino acid sequences selected from the group consisting of:    -   a) SEQ ID NOS: 70, 73, and 75; and    -   b) SEQ ID NOS: 71, 73, and 75.-   5. The antibody or antigen-binding portion of embodiment 1 or 3,    wherein the heavy chain of said antibody comprises a CDR3 comprising    SEQ ID NO: 67 or 68 and the light chain of said antibody comprises a    CDR3 comprising SEQ ID NO: 75.-   6. The antibody or antigen-binding portion according to embodiment 2    or 4, wherein said heavy chain CDR1, CDR2, and CDR3 and said light    chain CDR1, CDR2, and CDR3 respectively comprise amino acid    sequences selected from the group consisting of:    -   a) SEQ ID NOS: 60, 64, 67, 70, 73, and 75;    -   b) SEQ ID NOS: 60, 64, 67, 71, 73, and 75;    -   c) SEQ ID NOS: 60, 63, 67, 70, 73, and 75;    -   d) SEQ ID NOS: 60, 63, 67, 71, 73, and 75;    -   e) SEQ ID NOS: 61, 65, 68, 70, 73, and 75;    -   f) SEQ ID NOS: 61, 65, 68, 71, 73, and 75;    -   g) SEQ ID NOS: 77, 79, 67, 70, 73, and 75;    -   h) SEQ ID NOS: 77, 79, 67, 71, 73, and 75;    -   i) SEQ ID NOS: 78, 80, 68, 70, 73, and 75; and    -   j) SEQ ID NOS: 78, 80, 68, 71, 73, and 75.-   7. A monoclonal anti-human CCL20 antibody or an antigen-binding    portion thereof, wherein said antibody comprises a heavy chain whose    variable domain comprises an amino acid sequence selected from SEQ    ID NOS: 9-14.-   8. A monoclonal anti-human CCL20 antibody or an antigen-binding    portion thereof, wherein said antibody comprises a light chain whose    variable domain comprises SEQ ID NO: 15 or 16.-   9. The antibody or antigen-binding portion according to embodiment 7    or 8, wherein said heavy chain variable domain and said light chain    variable domain respectively comprise amino acid sequences selected    from the group consisting of:    -   a) SEQ ID NOS: 9 and 15;    -   b) SEQ ID NOS: 9 and 16;    -   c) SEQ ID NOS: 10 and 16;    -   d) SEQ ID NOS: 11 and 15;    -   e) SEQ ID NOS: 11 and 16;    -   f) SEQ ID NOS: 12 and 16;    -   g) SEQ ID NOS: 13 and 16; and    -   h) SEQ ID NOS: 14 and 16.-   10. A monoclonal anti-human CCL20 antibody whose heavy chain    comprises an amino acid sequence selected from the group consisting    of SEQ ID NOS: 1-6 without the signal sequence and SEQ ID NO: 108.-   11. A monoclonal anti-human CCL20 antibody whose heavy chain    comprises an amino acid sequence selected from the group consisting    of SEQ ID NOS: 1-6 without the signal sequence and SEQ ID NO: 108,    wherein said amino acid sequence lacks the C-terminal lysine.-   12. A monoclonal anti-human CCL20 antibody whose light chain    comprises an amino acid sequence selected from the group consisting    of SEQ ID NOS: 7 and 8 without the signal sequence and SEQ ID NOS:    110 and 112.-   13. The antibody according to any one of embodiments 10-12, wherein    said heavy chain and said light chain respectively comprise amino    acid sequences selected from the group consisting of:    -   a) SEQ ID NOS: 1 and 7;    -   b) SEQ ID NOS: 1 and 8;    -   c) SEQ ID NOS: 2 and 8;    -   d) SEQ ID NOS: 3 and 7;    -   e) SEQ ID NOS: 3 and 8;    -   f) SEQ ID NOS: 4 and 8;    -   g) SEQ ID NOS: 5 and 8;    -   h) SEQ ID NOS: 6 and 8;    -   i) SEQ ID NOS: 108 and 110; and    -   j) SEQ ID NOS: 108 and 112;    -   wherein said amino acid sequences lack signal sequences, if        present, and wherein SEQ ID NOS: 1-6 optionally lack the        C-terminal lysine.-   14. A monoclonal antibody whose heavy chain comprises SEQ ID NO: 108    and whose light chain comprises SEQ ID NO: 110.-   15. A monoclonal antibody whose heavy chain comprises SEQ ID NO: 108    without the C-terminal lysine and whose light chain comprises SEQ ID    NO: 110.-   16. A monoclonal antibody whose heavy chain comprises SEQ ID NO: 108    and whose light chain comprises SEQ ID NO: 112.-   17. A monoclonal antibody whose heavy chain comprises SEQ ID NO: 108    without the C-terminal lysine and whose light chain comprises SEQ ID    NO: 112.-   18. A monoclonal anti-human CCL20 antibody, or an antigen-binding    portion thereof, that binds to the same epitope of human CCL20 as    the monoclonal antibody or antigen-binding portion of any one of    embodiments 1-17.-   19. A monoclonal anti-human CCL20 antibody, or an antigen-binding    portion thereof, that competes for binding to human CCL20 with the    monoclonal antibody or antigen-binding portion of any one of    embodiments 1-17.-   20. A monoclonal anti-human CCL20 antibody, or an antigen-binding    portion thereof, that cross-competes for binding to human CCL20 with    the monoclonal antibody or antigen-binding portion of any one of    embodiments 1-17.-   21. The monoclonal antibody or antigen-binding portion of any one of    embodiments 1-12, wherein the antibody is a humanized antibody.-   22. The monoclonal antibody or antigen-binding portion of any one of    embodiments 1-6, wherein the framework regions of said heavy chain    utilize a IGHV1-46*03 human germline sequence, and wherein the    framework regions of said light chain utilize a IGKV1D-39*01 human    germline sequence.-   23. The monoclonal antibody of any one of embodiments 1-9 and 18-22,    wherein said antibody comprises a human IgG1, IgG2, IgG3, or IgG4    constant domain.-   24. A monoclonal anti-human CCL20 antibody, or an antigen-binding    portion thereof, whose heavy chain comprises a variable domain    comprising an amino acid sequence selected from the group consisting    of SEQ ID NOS: 39, 41, and 43.-   25. A monoclonal anti-human CCL20 antibody, or an antigen-binding    portion thereof, whose light chain comprises a variable domain    comprising an amino acid sequence selected from the group consisting    of SEQ ID NOS: 40, 42, and 44.-   26. The antibody or antigen-binding portion of embodiment 24 or 25,    wherein said heavy chain comprises an amino acid sequence selected    from the group consisting of SEQ ID NOS: 39, 41, and 43 and wherein    said light chain comprises an amino acid sequence selected from the    group consisting of SEQ ID NOS: 40, 42, and 44.-   27. The antibody of embodiment 26, wherein said heavy chain and said    light chain respectively comprise amino acid sequences selected from    the group consisting of:    -   a) SEQ ID NOS: 39 and 40;    -   b) SEQ ID NOS: 41 and 42; and    -   c) SEQ ID NOS: 43 and 44.-   28. A monoclonal anti-human CCL20 antibody, or an antigen-binding    portion thereof, wherein said antibody comprises a heavy chain    comprising SEQ ID NO: 39 and a light chain comprising SEQ ID NO: 40.-   29. The monoclonal antibody or antigen-binding portion of any one of    embodiments 24-28, wherein said antibody is a chimeric antibody.-   30. The antigen-binding portion of any one of embodiments 1-9,    18-22, and 24-28, wherein said portion is a single chain antibody,    Fv, Fab, Fab′, F(ab′)₂, Fd, single chain Fv molecule (scFv),    bispecific single chain Fv dimer, diabody, domain-deleted antibody    or single domain antibody (dAb).-   31. The monoclonal antibody or antigen-binding portion according to    any one of embodiments 1-30, wherein said antibody or    antigen-binding portion has one or more properties selected from the    group consisting of:    -   a) does not bind to human CCL16;    -   b) binds to cynomolgus or rhesus CCL20, but not to mouse or rat        CCL20;    -   c) has a binding affinity for human CCL20 of 70 pM or less using        a monovalent surface plasmon resonance assay;    -   d) has a binding affinity for human CCL20 of 12 pM or less using        a bivalent surface plasmon resonance assay;    -   e) has a binding affinity for human CCL20 greater than that of        human CCR6;    -   f) has a selectivity for human CCL20 over human CX3CL1, CXCL1,        CXCL2, CXCL4, CXCL8, CXCL9, CXCL10, CXCL12, CXCL13, CXCL16,        CCL1, CCL2, CCL3, CCL4, CCL5, CCL7, CCL11, CCL13, CCL16, CCL17,        CCL19, CCL21, CCL22, CCL24, CCL25, CCL27, CCL28, or XCL1;    -   g) reduces human CCL20-induced chemotaxis of CCR6+ cells with an        IC₅₀ of 1.7 nM or less;    -   h) reduces human CCL20-induced chemotaxis of CCR6+ cells in        vivo;    -   i) reduces human CCL20-induced chemotaxis of CCR6+ cells in        vitro;    -   j) reduces progression of arthritis symptoms in a subject;    -   k) reduces osteoporosis, bone erosion, or new bone formation in        a subject;    -   l) reduces cartilage oligomeric matrix protein (COMP) serum        levels in a subject;    -   m) reduces mRNA levels of RANKL, RANK, TRAP, or cathepsin K in a        subject;    -   n) reduces progression of atopic dermatitis in a subject; and    -   o) reduces progression of allergic contact dermatitis in a        subject.-   32. A monoclonal anti-human CCL20 antibody, or an antigen-binding    portion thereof, that binds to an epitope of human CCL20 comprising    one or more amino acid sequences selected from the group consisting    of:    -   a) residues 7-9 of SEQ ID NO: 84;    -   b) residues 10-19 of SEQ ID NO: 84; and    -   c) residues 20-22 of SEQ ID NO: 84.-   33. The monoclonal antibody or antigen-binding portion of embodiment    32, wherein said epitope comprises residues 7-9, 10-19, and 20-22 of    SEQ ID NO: 84.-   34. The monoclonal antibody or antigen-binding portion of embodiment    32, wherein said epitope further comprises one or more amino acid    sequences selected from the group consisting of:    -   a) residues 39-55 of SEQ ID NO: 84;    -   b) residues 56-67 of SEQ ID NO: 84; and    -   c) residues 61-70 of SEQ ID NO: 84.-   35. The monoclonal antibody or antigen-binding portion of embodiment    34, wherein said epitope comprises residues 7-9, 10-19, 20-22,    39-55, 56-67, and 61-70 of SEQ ID NO: 84.-   36. An isolated nucleic acid molecule encoding the heavy chain or an    antigen-binding portion thereof of an antibody or portion according    to any one of embodiments 1-35.-   37. An isolated nucleic acid molecule encoding the light chain or an    antigen-binding portion thereof of an antibody or portion according    to any one of embodiments 1-35.-   38. An isolated nucleic acid molecule encoding the heavy chain or an    antigen-binding portion thereof, and the light chain or an    antigen-binding portion thereof, of an antibody or portion according    to any one of embodiments 1-35.-   39. An isolated nucleic acid molecule encoding the heavy chain or an    antigen-binding portion thereof of a monoclonal anti-human CCL20    antibody, wherein said nucleic acid molecule comprises a nucleotide    sequence selected from the group consisting of SEQ ID NOS: 17-22,    25-30, and 109, or said nucleotide sequence without the sequence    encoding a signal sequence, if present.-   40. An isolated nucleic acid molecule encoding the light chain or an    antigen-binding portion thereof of a monoclonal anti-human CCL20    antibody, wherein said nucleic acid molecule comprises a nucleotide    sequence selected from the group consisting of SEQ ID NOS: 23, 24,    31, 32, 111, and 113, or said nucleotide sequence without the    sequence encoding a signal sequence, if present.-   41. The isolated nucleic acid molecule of embodiment 39 or 40,    wherein said nucleic acid molecule comprises a nucleotide sequence    selected from the group consisting of SEQ ID NOS: 17-22, 25-30, and    109, or said nucleotide sequence without the sequence encoding a    signal sequence if present; and wherein said nucleic acid molecule    further comprises a nucleotide sequence selected from the group    consisting of SEQ ID NOS: 23, 24, 31, 32, 111, and 113, or said    nucleotide sequence without the sequence encoding a signal sequence    if present.-   42. Use of (1) a nucleic acid sequence encoding the heavy chain or    an antigen-binding portion thereof, (2) a nucleic acid sequence    encoding the light chain or an antigen-binding portion thereof,    or (3) both, of an antibody or portion according to any one of    embodiments 1-35, as a medicament.-   43. A recombinant vector comprising (1) a nucleic acid sequence    encoding the heavy chain or an antigen-binding portion thereof, (2)    a nucleic acid sequence encoding the light chain or an    antigen-binding portion thereof, or (3) both, of an antibody or    portion according to any one of embodiments 1-35.-   44. A host cell comprising a first nucleic acid sequence encoding    the heavy chain or an antigen-binding portion thereof of an antibody    or portion according to any one of embodiments 1-35, said first    nucleic acid sequence operably linked to an expression control    element, and a second nucleic acid sequence encoding the light chain    or an antigen-binding portion thereof of said antibody or portion,    said second nucleic acid sequence operably linked to an expression    control element.-   45. A method of making an anti-human CCL20 antibody or an    antigen-binding portion thereof, comprising maintaining the host    cell of embodiment 44 under conditions appropriate for expression of    the antibody or portion.-   46. The method of embodiment 45, further comprising isolating the    antibody or portion.-   47. A composition comprising the monoclonal antibody or    antigen-binding portion of any one of embodiments 1-35 and a    pharmaceutically acceptable vehicle or carrier.-   48. A method for treating a subject in need thereof, comprising    administering to the subject an effective amount of the antibody or    antigen-binding portion of any one of embodiments 1-35 or the    composition of embodiment 47.-   49. A method for treating a condition in a subject in need thereof,    comprising administering to the subject an effective amount of the    antibody or antigen-binding portion of any one of embodiments 1-35    or the composition of embodiment 47.-   50. The method of embodiment 49, wherein said condition is a    CCR6-associated condition.-   51. The method of embodiment 49, wherein said condition is an    autoimmune or inflammatory condition.-   52. The method of embodiment 49, wherein said condition is    rheumatoid arthritis, psoriasis, atopic dermatitis, contact    dermatitis, Crohn's disease, inflammatory bowel disease, Grave's    disease, vitiligo, hyperthyroidism, chronic hepatitis, human    papillomavirus infection of the cervix, mycosis fungoides,    osteoporosis, or periodontal disease.-   53. A method for treating cancer, comprising administering to a    subject an effective amount of the antibody or antigen-binding    portion of any one of embodiments 1-35 or the composition of    embodiment 47.-   54. The method of embodiment 53, wherein said cancer is a B-cell    malignancy, breast adenocarcinoma, glioblastoma, hepatocellular    carcinoma, pancreatic adenocarcinoma, or thyroid papillary    carcinoma.-   55. A method for reducing CCL20-mediated chemotaxis of CCR6+ cells    in a subject in need thereof, comprising administering to the    subject the antibody or antigen-binding portion of any one of    embodiments 1-35 or the composition of embodiment 47.-   56. A method for reducing CCL20-mediated chemotaxis of CCR6+ cells    in vitro, using the antibody or antigen-binding portion of any one    of embodiments 1-35 or the composition of embodiment 47.-   57. A method for treating a condition in a subject, comprising    administering to the subject the antibody or antigen-binding portion    of any one of embodiments 1-35 or the composition of embodiment 47,    wherein said condition is selected from the group consisting of:    -   a) articular lesions of extremities distal to the elbow or knee;    -   b) erythema;    -   c) swelling;    -   d) increased cartilage oligomeric matrix protein (COMP) serum        levels;    -   e) increased mRNA levels of receptor activator for nuclear        factor κB ligand (RANKL), receptor activator for nuclear factor        κB (RANK), tartrate resistant acid phosphatase (TRAP), or        cathepsin K;    -   f) atopic dermatitis; and    -   g) allergic contact dermatitis.-   58. Use of the antibody or antigen-binding portion of any one of    embodiments 1-35 or the composition of embodiment 47 for the    manufacture of a medicament.-   59. Use of the antibody or antigen-binding portion of any one of    embodiments 1-35 or the composition of embodiment 47 as a    medicament.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 is a graph showing that CCR6 deficient mice are less sensitive tocollagen-induced arthritis than wild-type mice. Wild type, CCR6^(+/+)mice; Het, CCR6^(+/−) mice; Knockout, CCR6^(−/−) mice. See Example 1.

FIG. 2 is a graph showing that CCR6 deficient mice exhibit impairedinfiltration of T-cells (as indicated by CD3 levels) in collagen-inducedarthritic lesions in comparison to wild-type mice. Data shown aremeans±SEM from one experiment (n=7 mice per group). *p<0.05, **p<0.01.See Example 1.

FIG. 3 is a graph showing that CCR6 deficient mice exhibit impairedinfiltration of macrophages (as indicated by F4/80 levels) incollagen-induced arthritic lesions in comparison to wild-type mice. Datashown are means±SEM from one experiment (n=7 mice per group). **p<0.01.See Example 1.

FIG. 4 is a graph showing that in comparison to wild-type mice, CCR6deficient mice are resistant to increased ear thickness in adinitrofluorobenzene (DNFB)-induced allergic contact dermatitis model.WT, wild-type mice; KO, CCR6 deficient mice. See Example 1.

FIG. 5 is a graph showing that 2F5-5 MAb inhibits CCL20-inducedchemotaxis. See Example 2.

FIGS. 6A-C depict sequence alignments of variable domains of antibodiesof the invention (SEQ ID NOS: 9-16) with those of mouse anti-humanantibodies 36F7C10 (VH: SEQ ID NO: 39; VL: SEQ ID NO: 40) and 42G5B10(VH: SEQ ID NO: 43) and with germline sequences IGHV1-46*03 (SEQ ID NO:57), JH4 (SEQ ID NO: 58), and IGKV1D-39*01 (SEQ ID NO: 59). Kabat andChothia definitions of each CDR are indicated. ABM67212 (SEQ ID NO: 82)and BAH04867.1 (SEQ ID NO: 83) are human antibodies from which theframework regions of the shown humanized antibody chains were derived.See Example 3.

FIGS. 7A-C are tables depicting the inhibition of in vitro chemotaxis bydifferent combinations of humanized anti-human CCL20 antibody chains.Data represent three trials, except where indicated. See Example 4.

FIGS. 8A-C depict three independent trials performed to assess theinhibitory effect of the humanized HC2/LK3 anti-human CCL20 antibody.The HC2/LK3 antibody is shown to inhibit CCL20-induced chemotaxis.Values for IC₅₀, IC₉₀, and IC₉₅ are shown for each experiment. SeeExample 4.

FIGS. 9A-C depict a series of graphs showing that humanized antibodiesB) HC2/LK3 and C) HC2/LC3 demonstrate dose dependent inhibition of cellmigration comparable to that of A) mouse antibody 36F7C10 in atransendothelial migration assay. See Example 4.

FIGS. 10A and B depict Biacore™ sensograms demonstrating that the A)HC2/LC3 and B) HC2/LK3 humanized antibodies bind to human CCL20 in aconcentration-dependent manner. See Example 5.

FIGS. 11A-D demonstrate that anti-human CCL20 antibodies specificallybind to human CCL20. An ELISA assay with plate-bound CCL20 and otherchemokines (at 1 μg/ml) was used to detect binding of A) 36F7C10 andchimeric antibodies, and B) humanized HC2LK3 and HC2LC3 antibodies. C)An ELISA assay with His-tag anchored CCL20 was used to detect binding.D) Biacore™ experiments confirm that anti-human CCL20 antibodies bind tohuman CCL20, and exhibit negligible binding to CXCL4. See Example 6.

FIG. 12 shows an amino acid sequence alignment between a56-residueoverlap portion of CCL20 (SEQ ID NO: 114) and CCL16 (SEQ ID NO: 115).See Example 6.

FIGS. 13A and B are a series of graphs showing that A) chimeric and B)humanized anti-human CCL20 antibodies react specifically with CCL20 anddo not react with CCL16. See Example 6.

FIG. 14 shows amino acid sequence alignments between human (firstdisclosure is SEQ ID NO: 85 and second disclosure is residues 2-96 ofSEQ ID NO: 85), rhesus (SEQ ID NO: 86), cynomolgus (SEQ ID NO: 87), andmouse (SEQ ID NO: 88) CCL20 orthologs. See Example 7.

FIG. 15 is a schematic representation of anti-human CCL20 antibodydetection in ELISA assays. See Example 7.

FIGS. 16A-C depict a series of graphs demonstrating that A) chimeric andB), C) humanized anti-human CCL20 antibodies bind effectively to human,rhesus, and cynomolgus CCL20, but not to mouse CCL20. See Example 7.

FIGS. 17A and B depict graphs demonstrating that humanized and chimericanti-human CCL20 antibodies bind effectively to human, rhesus, andcynomolgus CCL20, but not to rat or mouse CCL20. The B) humanized and A)chimeric antibodies retain the binding specificity of A) mouse antibody36F7C10, from which they are derived. See Example 7.

FIGS. 18A and B show the results of epitope mapping of human CCL20 byhydrogen/deuterium exchange. This experiment utilized a variant of humanCCL20 (SEQ ID NO: 84) from R&D Systems in which the second to lastresidue is a D instead of the N shown in the wild-type sequence. A) Thedeuteration level of each residue of CCL20 is indicated at four timepoints (from top: 150 s, 500 s, 1500 s, and 5000 s). B) Structure ofhuman CCL20, indicating an epitope bound by antibodies of the invention.See Example 9.

FIG. 19 is a pair of graphs demonstrating that mouse CCR6-transducedB300 cells migrate toward both mouse and human CCL20. See Example 10.

FIG. 20 is a graph showing that mouse, chimeric, and humanizedanti-human CCL20 antibodies inhibit mouse T-cell migration toward humanCCL20 in vivo in a dose-dependent fashion. See Example 10.

FIG. 21 is a graph showing that hamster anti-mouse CCL 20 MAb 2F5-5reduces collagen-induced arthritis symptoms in mice. See Example 11.

FIG. 22 is a table of individual animal clinical scores demonstratingthat 2F5-5 MAb reduces progression of collagen-induced arthritissymptoms. See Example 11.

FIG. 23 is a graph depicting the grading of mouse paws by X-ray scoring.2F5-5 MAb is shown to reduce bone pathology in mice withcollagen-induced arthritis. Cont: control IgG. See Example 11.

FIG. 24 depicts examples of scored X-rays indicating bone pathology inthe paws of mice with collagen-induced arthritis. O: osteoporosis, E:bone erosion, N: new bone formation. See Example 11.

FIGS. 25A-C show measurements of a marker of cartilage destruction,serum cartilage oligomeric matrix protein (COMP), in a collagen-inducedarthritic mouse model. A) Calibrator data with a COMP standard. X axis,units/liter; Y axis, optical density at 450 nm. B) Plate template andraw data for mice treated with a hamster control IgG or 2F5-5 MAb. C)Data from individual animals demonstrates reduced COMP levels in animalstreated with 2F5-5 MAb compared to animals treated with control IgGantibodies. See Example 11.

FIG. 26 is a graph showing that 2F5-5 MAb decreases serum levels of COMPin mice. Ctrl IgG: isotype-matched antibody; CCL20 mAb: 2F5-5 MAb. SeeExample 11.

FIGS. 27A and B are graphs showing that 2F5-5 MAb reduces mRNAexpression of osteoclast markers A) RANKL and RANK, and B) TRAP andCathepsin K. Ctrl IgG: isotype-matched antibody; CCL20 mAb: 2F5-5MAb; Yaxis: quantitative PCR units. See Example 11.

FIG. 28 is a graph showing that 2F5-5 MAb has a prophylactic effect onglucose-6-phosphate isomerase-induced arthritis in mice. See Example 12.

FIG. 29 is a graph showing that 2F5-5 MAb suppresses the progression ofoxazolone-induced atopic dermatitis in mice. See Example 13.

FIG. 30 is a graph showing that 2F5-5 MAb inhibitsdinitrofluorobenzene-induced allergic contact dermatitis (as measured byear thickness) in mice. See Example 14.

DETAILED DESCRIPTION OF THE INVENTION

This invention is directed to antibodies that specifically bind to CCL20or a portion thereof (e.g., an antigenic portion thereof). The inventionis also directed to antigen-binding portions of said antibodies. In oneembodiment, the antibodies neutralize one or more activities of CCL20.An antibody is said to bind specifically to CCL20 if it does notsubstantially bind to non-CCL20 molecules. Substantial binding is, forexample, binding with a K_(D) of ≦100 nM, preferably ≦than 10 nM, 1 nM,100 pM, 50 pM, 40 pM, or 35 pM as determined by Biacore™ (in bivalentformat). In one embodiment, the antibodies or portions bind specificallyto human CCL20 or a portion thereof, to some sequence variants of humanCCL20 such as allelic variants, and may also cross-react with CCL20 fromother species. In one embodiment, the antibodies or portions havebinding specificity for a wild-type (also referred to as naturallyoccurring or endogenous) human CCL20. Unless otherwise indicated, “humanCCL20” refers to wild-type human CCL20. The amino acid sequence of awild-type human CCL20 with the signal sequence is shown in FIG. 14 (SEQID NO: 85). The amino acid sequence of a wild-type human CCL20 withoutthe signal sequence (residues 1-26 of SEQ ID NO: 85) is found in SEQ IDNO: 99 (see Table 18). FIG. 18 depicts a variant of human CCL20 withoutthe signal sequence (SEQ ID NO: 84) wherein the next to last residue isa D instead of the N as in the wild-type sequence (SEQ ID NO: 85). Insome embodiments, the antibodies or portions bind to wild-type humanCCL20, or human CCL20 wherein the next to last residue is a D instead ofthe N shown in the wild-type sequence, either sequence with or withoutthe signal sequence (e.g., SEQ ID NO: 84, 85, or 99). In a particularembodiment, the antibodies or portions bind specifically to human,rhesus, and cynomolgus CCL20 but do not bind to mouse or rat CCL20.

The antibodies and antigen-binding portions thereof described herein canbe purified and/or isolated using known techniques. Antibodies orportions that are “purified” or “isolated” have been separated away frommolecules (e.g., peptides) of their source of origin (e.g., thesupernatant of cells; in a mixture such as in a mixture of antibodies ina library; etc.), and include antibodies obtained by methods describedherein or other suitable methods. Isolated antibodies includesubstantially pure (e.g., essentially pure) antibodies, as well asantibodies produced by chemical synthesis, recombinant techniques and acombination thereof.

More specifically, the invention relates to anti-human CCL20 antibodies,antigen-binding portions (i.e., portions) of the antibodies, the lightchains of the antibodies, the heavy chains of the antibodies, andportions of these light chains or heavy chains. The invention alsorelates to antibodies lacking the heavy and/or light chain signalsequences and glycosylated antibodies. The invention also relates toprecursor antibodies, nonglycosylated antibodies, and antibodies whoseheavy and/or light chains comprise signal sequences. The invention alsorelates to nucleic acid molecules that encode any of the antibody heavychains and/or light chains, or portions thereof described herein; tovectors and host cells that comprise such nucleic acids; to methods ofproducing any of the antibody heavy and/or light chains or portionsthereof described herein; and to methods of using the antibodies,antibody chains, or portions.

The antibodies and antigen-binding portions thereof of this inventioncan be used to treat a subject in need thereof (e.g., a human patient)to reduce CCL20 binding to CCR6, CCL20-mediated inflammation, and/orCCL20-mediated chemotaxis of CCR6+ cells as needed.

Antibodies of the invention include traditional antibodies comprisingtwo heavy chains and two light chains. In some embodiments, one or moreof the heavy and/or light chains comprises a variable domain (alsoreferred to herein as a “variable region”) and a constant domain (alsoreferred to herein as a “constant region”). Complete variable domains,where present, comprise four framework regions (FRs) and threecomplementarity determining regions (CDRs), arranged, proceeding fromthe amino terminus, in the order FR1, CDR1, FR2, CDR2, FR3, CDR3, FR4.Visual inspection and sequence analysis can be carried out to identifythe CDR boundaries. For this invention, the CDR sequences are defined byusing the Kabat system (Kabat, E. A. et al., Sequences of Proteins ofImmunological Interest, Fifth Edition, U.S. Department of Health andHuman Services, U.S. Government Printing Office (1991)) and/or theChothia system (Chothia & Lesk, Canonical Structures for theHypervariable Regions of Immunoglobulins, J. Mol. Biol. 196:901-917(1987)), as indicated in, e.g., FIG. 6.

Embodiments of the present invention that comprise a human heavy chainconstant region may comprise a human constant region of any isotype,including IgG, IgM, IgA, IgD and IgE, in which the heavy chains are ofthe gamma (γ), mu (μ), alpha (α), delta (δ) or epsilon (ε) type,respectively, and any subclasses including IgG1, IgG2, IgG3, IgG4, IgA1and IgA2, in which the heavy chains are of the γ1, γ2, γ3, γ4, α1 and α2type, respectively. Embodiments comprising human light chains maycomprise a human kappa (κ) or a human lambda (λ) light chain.

As used herein, the term “antibody” refers to a complete antibody(comprising two full-length heavy chains and two full-length lightchains). The term “antigen-binding fragment” is used interchangeablyherein with the term “antigen-binding portion” unless otherwiseindicated. Antigen-binding portions of antibodies can be in the formatof, for example, single chain antibodies, Fv fragments, Fab fragments,Fab′ fragments, F(ab′)₂ fragments, Fd fragments, single chain Fvmolecules (scFv), bispecific single chain Fv dimers (PCT/US92/09665),diabodies, domain-deleted antibodies and single domain antibodies(dAbs). See, e.g., Jespers et al., Nature Biotechnology 22(9):1161-1165(2004)). Also within the invention are antigen-binding moleculescomprising a VH and/or a VL. In the case of a VH, the molecule may alsocomprise one or more of a CH1, hinge, CH2 and CH3 region.

Antibody portions can be produced by enzymatic cleavage or byrecombinant techniques. For instance, papain or pepsin cleavage can beused to generate Fab or F(ab′)₂ fragments, respectively. Antibodies alsocan be produced in a variety of truncated forms using antibody genes inwhich one or more stop codons have been introduced upstream of thenatural stop site. For example, a recombinant construct encoding theheavy chain of an F(ab′)₂ fragment can be designed to include DNAsequences encoding the CH₁ domain and hinge region of the heavy chain.

In another embodiment, a fusion antibody or immunoadhesin may be madethat comprises all or a portion of an anti-CCL20 antibody of theinvention linked to another polypeptide. In one embodiment, only thevariable domains of the anti-CCL20 antibody are linked to thepolypeptide. In another embodiment, the V_(H) domain of an anti-CCL20antibody is linked to a first polypeptide, while the V_(L) domain of ananti-CCL20 antibody is linked to a second polypeptide that associateswith the first polypeptide in a manner such that the V_(H) and V_(L)domains can interact with one another to form an antigen binding site.In yet another embodiment, the V_(H) domain is separated from the V_(L)domain by a linker such that the V_(H) and V_(L) domains can interactwith one another (see below under Single Chain Antibodies). TheV_(H)-linker-V_(L) antibody is then linked to the polypeptide ofinterest. In addition, fusion antibodies can be created in which two (ormore) single-chain antibodies are linked to one another, to create adivalent or polyvalent antibody on a single polypeptide chain, or tocreate a bispecific antibody.

To create a single chain antibody of the invention, the V_(H)- andV_(L)-encoding DNA fragments are operatively linked to another fragmentencoding a flexible linker, e.g., encoding the amino acid sequence(Gly₄-Ser)₃ (SEQ ID NO: 116), such that the V_(H) and V_(L) sequencescan be expressed as a contiguous single-chain protein, with the V_(L)and V_(H) domains joined by the flexible linker See, e.g., Bird et al.,Science 242:423-426 (1988); Huston et al., Proc. Natl. Acad. Sci. USA85:5879-5883 (1988); McCafferty et al., Nature 348:552-554 (1990); etc.In some embodiments, the single chain antibody is monovalent (only asingle V_(H) and V_(L) are used), bivalent (two V_(H) and V_(L) areused), or polyvalent (more than two V_(H) and V_(L) are used). Theinvention also contemplates bispecific or polyvalent antibodies thatbind specifically to human CCL20 and to another molecule.

In other embodiments, other modified antibodies may be prepared usinganti-CCL20 antibody-encoding nucleic acid molecules. For instance,“Kappa bodies” (Ill et al., Protein Eng. 10:949-57 (1997)), “Minibodies”(Martin et al., EMBO J. 13:5303-9 (1994)), “Diabodies” (Holliger et al.,Proc. Natl. Acad. Sci. USA 90:6444-6448 (1993)), or “Janusins”(Traunecker et al., EMBO J. 10:3655-3659 (1991) and Traunecker et al.,Int. J. Cancer (Suppl.) 7:51-52 (1992)) may be prepared using standardmolecular biological techniques following the teachings of thespecification.

In another aspect, the invention provides a variant of an antibodyexemplified herein, or an antigen-binding portion of said variantantibody, wherein said variant antibody binds to human CCL20specifically but differs in sequence from the exemplified antibody by 1,2, 3, 4, 5, 6, 7, 8, 9, 10, or more amino acid substitutions (forexample, in a CDR region, a FR region, and/or a constant domain).According to the invention, the variant antibody may be at least 80%, atleast 85%, at least 90%, at least 91%, at least 92%, at least 93%, atleast 94%, at least 95%, at least 96%, at least 97% at least 98% or atleast 99% identical to the reference antibody in the heavy chain, theheavy chain variable domain, the light chain, the light chain variabledomain, the six CDRs, or the eight FRs.

As used herein, sequence similarity for polypeptides, which is alsoreferred to as sequence identity, is typically measured using sequenceanalysis software, which matches similar sequences using measures ofsimilarity assigned to various substitutions, deletions and othermodifications, including conservative amino acid substitutions. Forinstance, the Genetics Computer Group (GCG) Sequence Analysis Packagecontains programs such as “Gap” and “Bestfit” which can be used withdefault parameters to determine sequence homology or sequence identitybetween closely related polypeptides, such as homologous polypeptidesfrom different species of organisms or between a wild type protein and amutein thereof. See, e.g., GCG Version 6.1. Polypeptide sequences canalso be compared with FASTA, a program in GCG Version 6.1, using defaultor recommended parameters. FASTA (e.g., FASTA2 and FASTA3) providesalignments and percent sequence identity of the regions of the bestoverlap between the query and search sequences (Pearson, MethodsEnzymol. 183:63-98 (1990); Pearson, Methods Mol. Biol. 132:185-219(2000)). Another algorithm used when comparing a sequence of theinvention to a database containing a large number of sequences fromdifferent organisms is the computer program BLAST, especially blastp ortblastn, using default parameters. See, e.g., Altschul et al., J. Mol.Biol. 215:403-410 (1990); Altschul et al., Nucleic Acids Res.25:3389-402 (1997); herein incorporated by reference.

According to the invention, one or more cysteines in the antibody, whichmay be chemically reactive, may be changed to another residue, such as,without limitation, alanine or serine. In one embodiment, anon-canonical cysteine is substituted. The substitution can be made in aCDR or framework region of a variable domain or in the constant domainof an antibody. In some embodiments, the cysteine is canonical. In someembodiments, potential proteolytic sites in the antibody are removed.Such sites may occur in a CDR or framework region of a variable domainor in the constant domain of an antibody. Substitution of cysteineresidues and removal of proteolytic sites may decrease the risk ofheterogeneity in the antibody product and thus increase its homogeneity.In some embodiments, asparagine-glycine pairs, which form potentialdeamidation sites, are eliminated by altering one or both of theresidues. In some embodiments, the antibody is deimmunized to reduce itsimmunogenicity. Techniques for reducing the immunogenicity of anantibody are well known in the art. See, e.g., PCT Publication Nos. WO98/52976 and WO 00/34317.

In some embodiments, the antibody has one or more conservative aminoacid substitutions when compared with an exemplified antibody of theinvention. A “conservative amino acid substitution” is one in which anamino acid residue is substituted by another amino acid residue having aside chain R group) with similar chemical properties (e.g., charge orhydrophobicity). In general, a conservative amino acid substitution willnot substantially change the functional properties of a protein. Incases where two or more amino acid sequences differ from each other byconservative substitutions, the percent sequence identity or degree ofsimilarity may be adjusted upwards to correct for the conservativenature of the substitution. Means for making this adjustment arewell-known to those of skill in the art. See e.g., Pearson, Methods Mol.Biol. 243:307-31 (1994).

Examples of groups of amino acids that have side chains with similarchemical properties include 1) aliphatic side chains: glycine, alanine,valine, leucine, and isoleucine; 2) aliphatic-hydroxyl side chains:serine and threonine; 3) amide-containing side chains: asparagine andglutamine; 4) aromatic side chains: phenylalanine, tyrosine, andtryptophan; 5) basic side chains: lysine, arginine, and histidine; 6)acidic side chains: aspartic acid and glutamic acid; and 7)sulfur-containing side chains: cysteine and methionine. Preferredconservative amino acids substitution groups are:valine-leucine-isoleucine, phenylalanine-tyrosine, lysine-arginine,alanine-valine, glutamate-aspartate, and asparagine-glutamine.Alternatively, a conservative replacement is any change having apositive value in the PAM250 log-likelihood matrix disclosed in Gonnetet al., Science 256:1443-45 (1992). A “moderately conservative”replacement is any change having a nonnegative value in the PAM250log-likelihood matrix.

In certain embodiments, amino acid substitutions to an antibody orantigen-binding portion of the invention are those which: (1) reducesusceptibility to proteolysis, (2) reduce susceptibility to oxidation,(3) alter binding affinity for forming protein complexes, for example,to enhance ADCC and CDC activity of the antibody, (4) confer or modifyother physicochemical or functional properties of such analogs, butstill retain specific binding to human CCL20, (5) remove C-terminallysine, and (6) add or remove glycosylation sites.

In one aspect, the invention provides a new and novel polypeptide thatis the heavy or light chain of an antibody of this invention, or that isa variable domain-containing portion of the heavy or light chain. Such apolypeptide is useful because it can partner with a light or heavyantibody chain, respectively, to form an anti-CCL20 antibody.

Described herein are novel humanized neutralizing anti-CCL20 antibodiescomprising the CDRs of novel mouse anti-human CCL20 antibodies, andantigen-binding portions of said humanized antibodies. The term“humanized anti-CCL20 antibody” as used herein refers to an antibodythat comprises one or more CDRs (CDR1, CDR2 and CDR3) of an anti-CCL20antibody of non-human origin, also referred to herein as the donorantibody (e.g., a mouse anti-CCL20 antibody), and at least a portionfrom a human sequence. The human antibody portion may be one or moreframework regions (e.g., all of the framework regions), and/or all orpart of a constant region. In some embodiments, the human sequenceframework region comprises a germline sequence, but may includenon-germline mutations. A CDR-grafted anti-CCL20 antibody in which thesix CDRs of a non-human anti-CCL20 antibody are grafted into a humanframework is an example of a humanized anti-CCL20 antibody of theinvention. See, e.g., Cabilly et al., U.S. Pat. No. 4,816,567; Cabillyet al., European Patent No. 0,125,023 B1; Boss et al., U.S. Pat. No.4,816,397; Boss et al., European Patent No. 0,120,694 B1; Neuberger, M.S. et al., WO 86/01533; Neuberger, M. S. et al., European Patent No.0,194,276 B1; Winter, U.S. Pat. No. 5,225,539; Winter, European PatentNo. 0,239,400 B1; Padlan, E. A. et al., European Patent Application No.0,519,596 A1. See also, Ladner et al., U.S. Pat. No. 4,946,778; Huston,U.S. Pat. No. 5,476,786; and Bird et al., Science 242:423-426 (1988)).In some embodiments, humanized antibodies are de-immunized antibodies.See, e.g., Can et al., U.S. Pat. No. 7,264,806, regarding de-immunizedantibodies that have been modified to reduce the number of potentialT-cell epitopes, thereby reducing the propensity for the antibody toelicit an immune response upon administration to a human.

In particular embodiments, the humanized antibody comprises one or morelight chain CDRs and/or one or more heavy chain CDRs of one or more ofthe following murine monoclonal anti-human CCL20 antibodies: 36F7C10,42G5B10, and 40-1C10B9, wherein the CDRs are identified according to theKabat system, the Chothia system, or any combination thereof. In someembodiments, the humanized antibody comprises all three heavy chain CDRsand all three light chain CDRs of antibody 36F7C10, 42G5B10, or40-1C10B9.

In another embodiment, the humanized antibodies have the bindingspecificity of a murine anti-human CCL20 antibody of the invention(e.g., specificity for human CCL20, the same or similar epitopicspecificity) and/or have a neutralizing activity. The humanizedantibodies can have the binding specificity, epitopic specificity,and/or neutralizing activity of a murine, chimeric, or humanizedanti-human CCL20 antibody described herein. For example, a humanizedantibody of the invention can compete with the murine, chimeric, orhumanized anti-human CCL20 antibody for binding to human CCL20, and/orit can have the neutralizing function of the murine, chimeric, orhumanized anti-human CCL20 antibody. In a particular embodiment, thehumanized antibody has the binding specificity, epitopic specificityand/or neutralizing activity of any one of mouse antibodies 36F7C10,42G5B10, and 40-1C10B9.

The human sequence portion of the humanized antibody (e.g., frameworkregion; constant region) can be from any suitable human antibody. Forexample, a human constant region or portion thereof in a humanized orchimeric antibody can be encoded by a human κ or λ light chain gene,and/or by a human γ (e.g., γ1, γ2, γ3, γ4), μ, α (e.g., α1, α2), δ or εheavy chain gene, including allelic variants. A particular humanconstant region isotype (e.g., IgG1; IgG2), variant or portion thereofcan be selected to tailor effector function. For example, a mutatedconstant region (i.e., a variant) can be incorporated into the antibodyto reduce binding to an Fc receptor and/or ability to fix complement.(See e.g., Winter et al., GB 2,209,757 B; Morrison et al., WO 89/07142;Morgan et al., WO 94/29351).

As used herein, the term “germline” refers to the nucleotide sequencesand amino acid sequences of the antibody genes and gene segments as theyare passed from parents to offspring via the germ cells. This germlinesequence is distinguished from the nucleotide sequences encoding aparticular antibody in a B cell, which has been altered by recombinationand hypermutation events during the course of affinity maturation. Anantibody that “utilizes” a particular germline has a nucleotide or aminoacid sequence that most closely aligns with the germline nucleotidesequence or with the amino acid sequence that it specifies as comparedto other germline sequences. Such antibodies may be encoded by orcomprise a sequence that is mutated compared with the germline sequence.

In some embodiments, the human framework has minimal variation fromgermline sequence, for example, less than 3, 4, 5, 6, 7, 8, 9, or 10acceptor framework residues have been replaced to improve one or moreproperties of the antibody. In some embodiments, acceptor frameworkresidues are replaced with donor framework residues, e.g., to improvebinding affinity (see, e.g., Queen et al., U.S. Pat. No. 5,530,101). Ina particular embodiment, a limited number of amino acids in theframework of a humanized antibody chain (e.g., 1, 2, 3, 4, 5, 6, 7, 8,9, or 10 amino acids) are chosen to be the same as the amino acids atthose positions in the donor sequence (i.e., “back-mutated”), ratherthan in the acceptor sequence, to increase the affinity of an antibodycomprising the humanized antibody chain for human CCL20.

Human framework regions (e.g., of the heavy and/or light chain variableregions) are preferably obtained or derived from a human germlinesequence having sequence similarity to the analogous or equivalentregion (e.g., heavy or light chain variable regions) of theantigen-binding region of the donor antibody (e.g., murine anti-CCL20antibody). Other sources of framework regions for human sequenceportions of a humanized antibody include human variable region consensussequences (See e.g., Kettleborough et al., Protein Engineering 4:773-783(1991); Carter et al., WO 94/04679; Carter U.S. Pat. No. 6,407,213)).For example, the region of the donor sequence of the antibody (e.g., thesequence of the variable region) used to obtain the nonhuman portion canbe compared to human sequences as described in Kabat, E. A. et al.Sequences of Proteins of Immunological Interest, Fifth Edition, U.S.Department of Health and Human Services, U.S. Government Printing Office(1991) to select a particular source of the human portions of thehumanized antibody, e.g., a source of the framework regions.

In one embodiment, the framework regions of the humanized antibodychains are obtained, or derived, from a human Ig variable region havingat least about 50%, at least about 55%, at least about 60%, at leastabout 65%, at least about 70%, at least about 75%, at least about 80%,at least about 85%, at least about 90% or at least about 95% overallsequence identity, with the variable region of the nonhuman donor. In aparticular embodiment, the framework regions of the humanized antibodychains are obtained or derived from human variable region frameworkregions having at least about 50%, at least about 55%, at least about60%, at least about 65%, at least about 70%, at least about 75%, atleast about 80%, at least about 85%, at least about 90%, or at leastabout 95% overall sequence identity with the framework regions of thevariable region of the nonhuman donor antibody.

In one embodiment, at least one of the framework regions (FR) of thehumanized antibody is obtained or derived from one or more chains of ahuman sequence. Thus, the FR can include a FR1 and/or FR2 and/or FR3and/or FR4 obtained or derived from one or more human sequenceantibodies (e.g., from a human antibody chain, from a human consensussequence).

It will be appreciated by one of skill in the art that in some casesresidues flanking the one or more CDRs of the murine anti-CCL20antibod(ies) may contribute, and in some cases, may be essential, eitherdirectly or indirectly, to function (e.g., binding). Accordingly, insome embodiments, one or more amino acids flanking one or more CDRs(e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more flanking amino acids) ofthe murine framework are also included in the humanized antibody.

In some embodiments, the human heavy chain framework regions of thehumanized antibodies of this invention utilize the human IGHV1-46*03germline sequence. In some embodiments, the human light chain frameworkregions of the humanized antibodies of this invention utilize the humanIGKV1D-39*01 germline sequence. Mutations (e.g., back mutations) mayoptionally be made in these FR regions, e.g., at one or more of theresidues as described in the Examples below to improve CCL20-bindingaffinity of the humanized antibody.

“Affinity” is a term of art that describes the strength of a bindinginteraction and typically refers to the overall strength of binding ofthe antibody to the antigen. The affinity of the antibody for theantigen is typically expressed as the binding affinity equilibriumconstant (K_(D)) of a particular antibody-antigen interaction.

In some embodiments, the antibody binds to human CCL20 with an affinity(K_(D); K_(D)=K_(off) (kd)/Kon (ka)) of 500 pM or less, 400 pM or less,300 pM or less, 200 pM or less, 100 pM or less, 90 pM or less, 80 pM orless, 70 pM or less, 60 pM or less, 50 pM or less, or 45 pM or less asdetermined by monovalent surface plasmon resonance; or 12 pM or less, 10pM or less, 8 pM or less, 6 pM or less, or 5 pM or less as determined bybivalent surface plasmon resonance. In some embodiments, the antibodybinds to human CCL20 with a k_(a) of 1000×10⁵ M⁻¹ sec⁻¹ or less, 900×10⁵M⁻¹ sec⁻¹ or less, 800×10⁵M⁻¹ sec⁻¹ or less, 700×10⁵ M⁻¹ sec⁻¹ or less,600×10⁵ M⁻¹ sec⁻¹ or less, 500×10⁵ M⁻¹ sec⁻¹ or less, 400×10⁵ M⁻¹ sec⁻¹or less, 300×10⁵ M⁻¹ sec⁻¹ or less, 240×10⁵M⁻¹ sec⁻¹ or less, 200×10⁵M⁻¹ sec⁻¹ or less, 190×10⁵ M⁻¹ sec⁻¹ or less, 180×10⁵ M⁻¹ sec⁻¹ or less,170×10⁵ M⁻¹ sec⁻¹ or less, 160×10⁵ M⁻¹ sec⁻¹ or less, or 150×10⁵ M⁻¹sec⁻¹ or less, as determined by monovalent surface plasmon resonance(Biacore™). In some embodiments, the antibody binds to human CCL20 witha k_(d) of 1000×10⁻⁵ sec⁻¹ or less, 900×10⁻⁵ sec⁻¹ or less, 800×10⁻⁵sec⁻¹ or less, 700×10⁻⁵ sec⁻¹ or less, 600×10⁻⁵ sec⁻¹ or less, 500×10⁻⁵sec⁻¹ or less, 400×10⁻⁵ sec⁻¹ or less, 300×10⁻⁵ sec⁻¹ or less, 240×10⁻⁵sec⁻¹ or less, 200×10⁻⁵ sec⁻¹ or less, 190×10⁻⁵ sec⁻¹ or less, 180×10⁻⁵sec⁻¹ or less, 170×10⁻⁵ sec⁻¹ or less, 160×10⁻⁵ sec⁻¹ or less, 150×10⁻⁵sec⁻¹ or less, 140×10⁻⁵ sec⁻¹ or less, 130×10⁻⁵ sec⁻¹ or less, 120×10⁻⁵sec⁻¹ or less, 100×10⁻⁵ sec⁻¹ or less, 90×10⁻⁵ sec⁻¹ or less, 80×10⁻⁵sec⁻¹ or less, 70×10⁻⁵ sec⁻¹ or less, or 65×10⁻⁵ sec⁻¹ or less asdetermined by monovalent surface plasmon resonance (Biacore™).

As is apparent to one of skill in the art, a variety of methods can beused to confirm that antibodies and antigen-binding portions thereofproduced according to methods provided herein and known in the art havethe requisite specificity (e.g., binding specificity, epitopicspecificity). For example, the binding function of a humanizedanti-CCL20 antibody or portion of the invention having bindingspecificity for human CCL20 can be detected using any suitable method,e.g., assays which monitor formation of a complex between the humanizedantibody or portion and human CCL20 (or, e.g., a peptide having an aminoacid sequence of CCL20 or a solid support comprising human CCL20).

The ability of an antibody or an antigen-binding portion thereof of theinvention (e.g., a humanized antibody or portion of the invention) tobind to the same epitope on human CCL20 as a particular murine,chimeric, or humanized monoclonal antibody disclosed herein, or to bindto an epitope on human CCL20 which overlaps with the epitope on humanCCL20 to which a particular murine, chimeric, or humanized monoclonalantibody disclosed herein binds, can be readily determined using avariety of techniques known to those of skill in the art, includinge.g., competitive binding assays. These may involve the use of a labeledform of said particular antibody, and a measurement of the binding ofthat labeled antibody to human CCL20 in the presence and in the absenceof an antibody of the invention.

An “epitope” as used herein includes any protein determinant capable ofspecific binding to an antibody. Methods for characterizing the epitopeto which an antibody binds are known in the art. One method ofcharacterizing an epitope bound by an anti-CCL20 antibody of theinvention is described in Example 9. Once a desired epitope on anantigen is determined, it is possible to generate antibodies to thatepitope, e.g., using the techniques described in the present invention.Alternatively, during the discovery process, the generation andcharacterization of antibodies may elucidate information about desirableepitopes. From this information, it is then possible to competitivelyscreen antibodies for binding to the same epitope. For example, theskilled worker may conduct competition studies to find antibodies thatcompetitively bind with one another, i.e., antibodies that compete forbinding to the antigen.

In one embodiment, to determine if a test antibody or antigen-bindingportion thereof binds to the same or overlapping epitope as a humanizedantibody of this invention, one allows the anti-CCL20 antibody of theinvention to bind to CCL20 under saturating conditions and then measuresthe ability of the test antibody to bind to CCL20. If the test antibodyis able to bind to CCL20 at the same time as the reference anti-CCL20antibody, then the test antibody may bind to a different epitope thanthe reference anti-CCL20 antibody. However, if the test antibody is notable to bind to CCL20 at the same time, then the test antibody may bindto the same epitope, an overlapping epitope, or an epitope that is inclose proximity to the epitope bound by the anti-CCL20 antibody of theinvention. This experiment can be performed using, e.g., ELISA, RIA,Biacore™, or flow cytometry. To test whether an anti-CCL20 antibodycross-competes with another anti-CCL20 antibody, one may use thecompetition method described above in two directions, i.e., determiningif the reference antibody blocks the test antibody and vice versa. Insome embodiments, the experiment is performed using Biacore™

Epitope binning also can be useful to characterize the antibodies ofthis invention. The term “binning” refers to a method to groupantibodies based on their antigen binding characteristics. A highthroughput process for “binning” antibodies based upon theircross-competition is described in International Patent Application No.WO 03/48731. The “epitope binning” can be investigated by allowing anunlabeled form of an anti-CCL20 antibody “A” to bind to a syntheticpeptide corresponding to the sequence of CCL20 or to CCL20 positivecells. Subsequently a labeled second anti-CCL20 antibody “B” is addedand one can assess the amount of labeled antibody that can bind relativeto a control sample where the cells or synthetic peptide have not beenexposed previously to anti-CCL20 antibody “A.” Alternatively, anti-CCL20antibodies “A” and “B” can both be labeled with different fluorochromesor chemicals enabling detection, and one can measure the quantities ofboth labeled antibodies that can engage the CCL20 peptide at the sametime using a device capable of detecting the label or measure theamounts of both antibodies that simultaneously engage CCL20 positivecells by flow cytometry. Biacore™ and Octet technologies enable one toinvestigate the competitive binding of unlabelled forms of antibodies.This use of unlabelled forms of antibodies is desired as the chemicalmodification of some antibodies can compromise the binding activity. Seealso the technology described in Jia et al., J. Immunol. Methods288:91-98 (2004), which is useful in performing epitope binning as well.

Also provided herein are portions of the anti-CCL20 antibodies of theinvention, such as light chains, heavy chains, and portions of light andheavy chains. These antibody portions can be obtained or derived fromantibodies (e.g., by reduction and/or cleavage), or produced orexpressed by nucleic acids encoding a portion of an antibody or chainthereof having the desired property (e.g., binds human CCL20, sequencesimilarity). They can also be prepared by e.g., de novo synthesis of therelevant portion. Humanized antibodies comprising the desired portions(e.g., antigen-binding region, CDR, FR, C region) of human and nonhumanorigin can be produced using synthetic and/or recombinant nucleic acidsto prepare constructs (e.g., cDNA) encoding the desired humanized chain.For example, to prepare a portion of an antibody (e.g., a portion of achain), one or more stop codons can be introduced at the desiredposition in the nucleic acid sequence. Nucleic acid (e.g., DNA)sequences coding for humanized variable regions can be constructed usingPCR mutagenesis methods to alter existing DNA sequences (see e.g.,Kamman et al., Nucl. Acids Res. 17:5404 (1989)). PCR primers coding forthe new CDRs can be hybridized to a DNA template of a previouslyhumanized variable region which is based on the same, or a very similar,human variable region (Sato et al., Cancer Research 53:851-856 (1993)).If a similar DNA sequence is not available for use as a template, anucleic acid comprising a sequence encoding a variable region sequencecan be constructed from synthetic oligonucleotides (see e.g., Kolbinger,Protein Engineering 8:971-980 (1993)). A sequence encoding a signalpeptide (“signal sequence”) can also be incorporated into the nucleicacid (e.g., on synthesis, upon insertion into a vector). If a signalpeptide sequence is unavailable (e.g., not typically present), a signalpeptide sequence from another antibody can be used (see, e.g.,Kettleborough, Protein Engineering 4:773-783 (1991)). Using thesemethods, methods described herein or other suitable methods, variantscan readily be produced.

As used herein, the acronym “mAb” (or “MAb”) refers to a monoclonalantibody, which may be, e.g., an antibody synthesized by a clonalpopulation of cells or a humanized antibody. A clonal population thatproduces a monoclonal antibody can be a clonal population ofimmortalized cells. In some embodiments, the immortalized cells in theclonal population are hybrid cells—hybridomas—typically produced by thefusion of individual B lymphocytes from an immunized animal withindividual cells from a lymphocytic tumour.

The invention relates in part to a humanized antibody or antigen-bindingportion thereof that has binding specificity for human CCL20 andcomprises a humanized light chain and a humanized heavy chain and/orportions thereof. In one embodiment, the humanized antibody comprises alight chain comprising one or more CDRs (e.g., all three CDRs) of SEQ IDNO: 7 and a heavy chain comprising one or more CDRs (e.g., all threeCDRs) of SEQ ID NO: 1; a light chain comprising one or more CDRs (e.g.,all three CDRs) of SEQ ID NO: 7 and a heavy chain comprising one or moreCDRs (e.g., all three CDRs) of SEQ ID NO: 2; a light chain comprisingone or more CDRs (e.g., all three CDRs) of SEQ ID NO: 7 and a heavychain comprising one or more CDRs (e.g., all three CDRs) of SEQ ID NO:3; a light chain comprising one or more CDRs (e.g., all three CDRs) ofSEQ ID NO: 7 and a heavy chain comprising one or more CDRs (e.g., allthree CDRs) of SEQ ID NO: 4; a light chain comprising one or more CDRs(e.g., all three CDRs) of SEQ ID NO: 7 and a heavy chain comprising oneor more CDRs (e.g., all three CDRs) of SEQ ID NO: 5; a light chaincomprising one or more CDRs (e.g., all three CDRs) of SEQ ID NO: 7 and aheavy chain comprising one or more CDRs (e.g., all three CDRs) of SEQ IDNO: 6; a light chain comprising one or more CDRs (e.g., all three CDRs)of SEQ ID NO: 8 and a heavy chain comprising one or more CDRs (e.g., allthree CDRs) of SEQ ID NO: 1; a light chain comprising one or more CDRs(e.g., all three CDRs) of SEQ ID NO: 8 and a heavy chain comprising oneor more CDRs (e.g., all three CDRs) of SEQ ID NO: 2; a light chaincomprising one or more CDRs (e.g., all three CDRs) of SEQ ID NO: 8 and aheavy chain comprising one or more CDRs (e.g., all three CDRs) of SEQ IDNO: 3; a light chain comprising one or more CDRs (e.g., all three CDRs)of SEQ ID NO: 8 and a heavy chain comprising one or more CDRs (e.g., allthree CDRs) of SEQ ID NO: 4; a light chain comprising one or more CDRs(e.g., all three CDRs) of SEQ ID NO: 8 and a heavy chain comprising oneor more CDRs (e.g., all three CDRs) of SEQ ID NO: 5; or a light chaincomprising one or more CDRs (e.g., all three CDRs) of SEQ ID NO: 8 and aheavy chain comprising one or more CDRs (e.g., all three CDRs) of SEQ IDNO: 6.

In one embodiment, a humanized antibody of the invention comprises heavychain (H)—CDR1, H-CDR2, H-CDR3, light chain (L)-CDR1, L-CDR2, and L-CDR3whose amino acid sequences are:

a) SEQ ID NOS: 60, 64, 67, 70, 73, and 75, respectively;

b) SEQ ID NOS: 60, 64, 67, 71, 73, and 75, respectively;

c) SEQ ID NOS: 60, 63, 67, 70, 73, and 75, respectively;

d) SEQ ID NOS: 60, 63, 67, 71, 73, and 75, respectively;

e) SEQ ID NOS: 61, 65, 68, 70, 73, and 75, respectively;

f) SEQ ID NOS: 61, 65, 68, 71, 73, and 75, respectively;

g) SEQ ID NOS: 77, 79, 67, 70, 73, and 75, respectively;

h) SEQ ID NOS: 77, 79, 67, 71, 73, and 75, respectively;

i) SEQ ID NOS: 78, 80, 68, 70, 73, and 75, respectively; and

j) SEQ ID NOS: 78, 80, 68, 71, 73, and 75, respectively.

In another embodiment, a humanized antibody of this invention comprisesan H-CDR3 whose sequence is SEQ ID NO: 67 or 68. In certain embodiments,a humanized antibody of this invention comprises H-CDR3 and L-CDR3 whosesequences are SEQ ID NOS: 67 and 75, respectively; or SEQ ID NOS: 68 and75, respectively.

In another embodiment, the humanized antibody has binding specificityfor human CCL20 and comprises a light chain comprising one or more CDRsselected from the group consisting of SEQ ID NOS: 70 or 71; 73; and 75,or a combination thereof; and a heavy chain comprising one or more CDRsselected from the group consisting of SEQ ID NOS: 60, 61, 77, or 78; 63,64, 65, 79, or 80; and 67 or 68; or a combination thereof.

In another embodiment, the humanized antibody that has a bindingspecificity for human CCL20 comprises a light chain comprising one ormore CDRs (e.g., all three CDRs) of SEQ ID NO: 7, 8, 110, or 112, and aheavy chain comprising one or more CDRs (e.g., all three CDRs) of SEQ IDNO: 1, 2, 3, 4, 5, 6, or 108.

The invention also relates to a humanized antibody light chain of thehumanized antibody described herein. In one embodiment, the humanizedantibody light chain comprises one or more CDRs selected from the groupconsisting of 70 or 71; 73; and 75, or a combination thereof. Forexample, the humanized antibody has L-CDR1, L-CDR2, and L-CDR3 whoseamino acid sequences are 70, 73, and 75 respectively; or 71, 73, and 75,respectively.

The invention also relates to a humanized antibody heavy chain of thehumanized antibody described herein. In one embodiment, the humanizedantibody heavy chain comprises one or more CDRs selected from the groupconsisting of 60, 61, 77, or 78; 63, 64, 65, 79, or 80; and 67 or 68; ora combination thereof. For example, the humanized antibody has H-CDR1,H-CDR2, and H-CDR3 whose amino acid sequences are:

a) SEQ ID NOS: 60, 63, and 67;

b) SEQ ID NOS: 60, 64, and 67;

c) SEQ ID NOS: 61, 65, and 68;

d) SEQ ID NOS: 77, 79, and 67; or

e) SEQ ID NOS: 78, 80, and 68.

In one embodiment, a humanized antibody of this invention comprises alight chain comprising a variable domain (V_(L)) sequence of SEQ ID NO:15 or 16. In a related embodiment, the humanized antibody comprises alight chain whose amino acid sequence comprises or consists of one ofSEQ ID NOS: 7, 8, 110, and 112. In one embodiment, the humanizedantibody comprises a light chain whose amino acid sequence comprises orconsists of SEQ ID NO: 7 or 8 without the signal sequence.

In one embodiment, a humanized antibody of this invention comprises aheavy chain comprising a variable domain (V_(H)) sequence of one of SEQID NOS: 9-14. In a related embodiment, the humanized antibody comprisesa heavy chain whose amino acid sequence comprises or consists of one ofSEQ ID NOS: 1-6 and 108. In one embodiment, the humanized antibodycomprises a heavy chain whose amino acid sequence comprises or consistsof one of SEQ ID NOS: 1-6 without the signal sequence, and optionallywithout the C-terminal lysine. In one embodiment, the humanized antibodycomprises a heavy chain whose amino acid sequence comprises or consistsof SEQ ID NO: 108 without the C-terminal lysine.

In some embodiments, a humanized antibody of this invention comprises aV_(H) and a V_(L) whose amino acid sequences comprise or consist of

a) SEQ ID NO: 9 and SEQ ID NO: 15;

b) SEQ ID NO: 10 and SEQ ID NO: 15;

c) SEQ ID NO: 11 and SEQ ID NO: 15;

d) SEQ ID NO: 12 and SEQ ID NO: 15;

e) SEQ ID NO: 13 and SEQ ID NO: 15;

f) SEQ ID NO: 14 and SEQ ID NO: 15;

g) SEQ ID NO: 9 and SEQ ID NO: 16;

h) SEQ ID NO: 10 and SEQ ID NO: 16;

i) SEQ ID NO: 11 and SEQ ID NO: 16;

j) SEQ ID NO: 12 and SEQ ID NO: 16;

k) SEQ ID NO: 13 and SEQ ID NO: 16; or

l) SEQ ID NO: 14 and SEQ ID NO: 16.

In one embodiment, a humanized antibody of this invention comprises alight chain (LC) and a heavy chain (HC) whose amino acid sequencescomprise or consist of

a) SEQ ID NO: 1 and SEQ ID NO: 7;

b) SEQ ID NO: 2 and SEQ ID NO: 7;

c) SEQ ID NO: 3 and SEQ ID NO: 7;

d) SEQ ID NO: 4 and SEQ ID NO: 7;

e) SEQ ID NO: 5 and SEQ ID NO: 7;

f) SEQ ID NO: 6 and SEQ ID NO: 7;

g) SEQ ID NO: 1 and SEQ ID NO: 8;

h) SEQ ID NO: 2 and SEQ ID NO: 8;

i) SEQ ID NO: 3 and SEQ ID NO: 8;

j) SEQ ID NO: 4 and SEQ ID NO: 8;

k) SEQ ID NO: 5 and SEQ ID NO: 8;

l) SEQ ID NO: 6 and SEQ ID NO: 8,

m) SEQ ID NO: 108 and SEQ ID NO: 110; and

n) SEQ ID NO: 108 and SEQ ID NO: 112;

wherein the amino acid sequences lack the signal sequence, if present,and wherein SEQ ID NOS: 1-6 and SEQ ID NO: 108 optionally lack theC-terminal lysine.

The present invention provides any combination comprising an exemplifiedhumanized heavy chain or an antigen-binding portion thereof, and anexemplified humanized light chain or an antigen-binding portion thereof,of the invention; in other words, the heavy and light chains can be“mixed and matched.” It is understood that any such combination islikely to retain binding to human CCL20 as well as chemotaxisneutralizing activity. These functional properties can readily be testedusing methods described herein. Indeed, FIG. 7 demonstrates theneutralizing activity of several combinations of exemplified humanizedheavy and light chains of the invention.

This invention also provides anti-human CCL20 antibodies orantigen-binding portions thereof that bind to the same epitope as,and/or compete or cross-compete with, an antibody exemplified herein.These antibodies can be, for example, humanized, chimeric, or mouseantibodies. For example, the invention provides anti-human CCL20antibodies and portions that bind to the same epitope as, and/or competeor cross-compete with, one of mouse anti-CCL20 antibodies 36F7C10,40-1C10B9, and 42G5B10, or humanized or chimeric versions of these mouseantibodies. The ability of an antibody to bind to the same epitope as,or compete or cross-compete with, a reference antibody, can bedetermined as described herein. By way of nonlimiting example, anyantibody or portion comprising the three heavy chain CDRs and the threelight chain CDRs from mouse antibody 36F7C10 would be expected to bindto the same epitope as, compete with, and cross-compete with mouseantibody 36F7C10. Such antibodies may include, e.g., antibodies whoseheavy chain comprises any one of SEQ ID NOS: 9-11 and whose light chaincomprises SEQ ID NO: 15 or 16. In some embodiments, such antibodies mayfurther include, e.g., antibodies whose heavy chain comprises any one ofSEQ ID NOS: 12-14 and whose light chain comprises SEQ ID NO: 15 or 16.

If desired, for example, for diagnostic or assay purposes (e.g., imagingto allow, for example, monitoring of therapies), the humanized antibody(or an antigen-binding portion thereof) can comprise a detectable label.Suitable detectable labels and methods for labeling a humanized antibodyor antigen-binding portion thereof are well known in the art. Suitabledetectable labels include, for example, a radioisotope (e.g., asIndium-111, Technnetium-99m or Iodine-131), positron emitting labels(e.g., Fluorine-19), paramagnetic ions (e.g., Gadlinium (III), Manganese(II)), an epitope label (tag), an affinity label (e.g., biotin, avidin),a spin label, an enzyme, a fluorescent group or a chemiluminescentgroup. When labels are not employed, complex formation (e.g., between ahumanized antibody and human CCL20) can be determined by surface plasmonresonance, ELISA, FACS, or other suitable methods.

Anti-CCL20 antibodies or antigen-binding portions thereof used in theinvention also may be conjugated, for example, via chemical reactions orgenetic modifications, to other moieties (e.g., pegylation moieties)that improve the antibodies' pharmacokinetics such as half-life. In someembodiments, the anti-CCL20 antibodies used in this invention can belinked to a suitable cytokine, e.g., via chemical conjugation or geneticmodifications (e.g., appending the coding sequence of the cytokine inframe to an antibody coding sequence, thereby creating anantibody:cytokine fusion protein).

The invention also relates to immunoconjugates in which the humanizedantibody (or an antigen-binding portion thereof) of the invention iscoupled to another therapeutic agent, such as a bioactive compound(e.g., a cytokine, a superantigen, a cytotoxic agent or a toxin). Forexample, the humanized antibody that has binding specificity for humanCCL20 (or an antigen binding portion thereof) can be coupled to abiological protein, a molecule of plant or bacterial origin (orderivative thereof), an interleukin-2 antibody or diptheria toxinantibodies.

As described herein, mouse monoclonal antibodies having bindingspecificity for human CCL20 have been produced. Humanized and chimericantibodies of this invention can be derived from the mouse monoclonalantibodies of this invention. That is, in some embodiments, humanizedand chimeric anti-CCL20 antibodies of the invention comprise sequencestaken from a mouse monoclonal antibody of the invention, such as one ormore CDR sequences (e.g., all six CDR sequences) or one or more variabledomains (e.g., the heavy chain variable domain and the light chainvariable domain).

As used herein, the term “mouse monoclonal antibody” refers to anantibody containing light chain CDRs (L-CDR1, L-CDR2 and L-CDR3) andheavy chain CDRs (H-CDR1, H-CDR2 and H-CDR3) of a murine antibody, andframework and constant regions of murine origin.

The invention relates to the mouse monoclonal antibodies describedherein, as well as antigen-binding portions of the mouse monoclonalantibodies, the light chains of the mouse monoclonal antibodies, theheavy chains of the mouse monoclonal antibodies, and portions of theseheavy and light chains. In a particular embodiment, the mouse monoclonalantibody is 36F7C10, 40-1C10B9, or 42G5B 10. The invention relates tomouse monoclonal antibodies lacking the heavy and light chain signalsequences and mouse monoclonal antibodies that are glycosylated. Theinvention also relates to precursor antibodies, nonglycosylatedantibodies, and antibodies whose heavy and/or light chains comprisesignal sequences. The invention also relates to nucleic acid moleculesthat encode any of the above mouse antibody heavy chains, light chains,or portions thereof; to vectors and host cells that comprise suchnucleic acids; to methods of producing any of the above mouse heavy orlight chains or portions thereof; and to methods of using the mouseantibodies or antigen-binding portions thereof.

The binding function of a mouse monoclonal antibody or antigen-bindingportion thereof having binding specificity for human CCL20 can bedetected using any suitable method, for example using assays whichmonitor formation of a complex between a mouse monoclonal antibody orportion and human CCL20 (or, e.g., a peptide having an amino acidsequence of CCL20 or a solid support comprising human CCL20).

Also provided herein are portions of the murine antibodies which includelight chains, heavy chains and portions of light and heavy chains. Theseantibody portions can be obtained or derived, e.g., by means describedherein for humanized antibody portions.

In one embodiment, a mouse monoclonal antibody of this inventioncomprises a light chain comprising SEQ ID NO: 40, 42, or 44 and furthercomprises a heavy chain comprising SEQ ID NO: 39, 41, or 43. In acertain embodiment, the mouse monoclonal antibody comprises a lightchain comprising SEQ ID NO: 40 and a heavy chain comprising SEQ ID NO:39; a light chain comprising SEQ ID NO: 42 and a heavy chain comprisingSEQ ID NO: 41; or a light chain comprising SEQ ID NO: 44 and a heavychain comprising SEQ ID NO: 43.

In another embodiment, the invention also relates to a mouse monoclonalantibody that has binding specificity for human CCL20, comprising thelight chain variable region in a sequence selected from the groupconsisting of SEQ ID NOS: 40, 42, and 44; and a heavy chain variableregion in a sequence selected from the group consisting of SEQ ID NOS:39, 41, and 43.

The invention also relates to a mouse monoclonal antibody whose lightchain comprises the variable region in SEQ ID NO: 40, 42, or 44.

The invention also relates to a mouse monoclonal antibody whose heavychain comprises the variable region in SEQ ID NO: 39, 41, or 43.

If desired, for example, for diagnostic or assay purposes (e.g.,imaging), the mouse monoclonal antibody or antigen binding portionthereof can comprise a detectable label e.g., as described herein forhumanized antibodies. All suitable methods and techniques describedherein for humanized antibodies of this invention can also be used formouse monoclonal antibodies of the invention.

As described herein, chimeric antibodies having binding specificity forhuman CCL20 have been produced. As used herein, the term “chimericantibody” refers to a recombinant protein that contains the variabledomains of an antibody derived from one species while the constantdomains of the antibody are derived from a different species. In oneembodiment, the chimeric antibody with binding specificity for humanCCL20 comprises variable domains from a mouse anti-human CCL20monoclonal antibody. In one embodiment, the chimeric antibody withbinding specificity for human CCL20 comprises constant domains from ahuman antibody. In a particular embodiment, the chimeric antibodycomprises variable domains from a mouse anti-human CCL20 monoclonalantibody and constant domains from a human antibody.

The invention relates to the chimeric antibodies described herein, aswell as antigen-binding portions of the chimeric antibodies, the lightchains and heavy chains of the chimeric antibodies, and portions ofthese light and heavy chains. The invention relates to chimericantibodies lacking the heavy and light chain signal sequences andchimeric antibodies that are glycosylated. The invention also relates toprecursor antibodies, nonglycosylated antibodies, and antibodies whoseheavy and/or light chains comprise signal sequences. The invention alsorelates to nucleic acid molecules that encode any of the above chimericantibody heavy chains, light chains, or portions thereof; to vectors andhost cells that comprise such nucleic acids; to methods of producingthese any of the above chimeric antibody heavy or light chains orportions thereof; and to methods of using the chimeric antibodies.

The binding function of a chimeric antibody having binding specificityfor human CCL20 can be detected using any suitable method, for exampleusing assays which monitor formation of a complex between a chimericantibody and human CCL20 (or, e.g., a peptide having an amino acidsequence of CCL20 or a solid support comprising human CCL20).

Also provided herein are portions of the chimeric antibodies whichinclude light chains, heavy chains and portions of light and heavychains. These antibody portions can be obtained or derived, e.g., bymeans described herein for humanized antibody portions.

In one embodiment, a chimeric antibody of this invention comprises thelight chain variable region of SEQ ID NO: 40 and the heavy chainvariable region of SEQ ID NO: 39; the light chain variable region of SEQID NO: 42 and the heavy chain variable region of SEQ ID NO: 41; or thelight chain variable region of SEQ ID NO: 44 and the heavy chainvariable region of SEQ ID NO: 43.

The invention also relates to a chimeric antibody that has bindingspecificity for human CCL20, comprising a light chain variable regionsequence selected from the group consisting of: the light chain variableregion in SEQ ID NO: 40, 42, or 44; and further comprising a heavy chainvariable region sequence selected from the group consisting of: theheavy chain variable region in SEQ ID NO: 39, 41, or 43.

The invention also relates to a chimeric light chain comprising thevariable region in SEQ ID NO: 40, 42, or 44.

The invention also relates to a chimeric heavy chain comprising thevariable region in SEQ ID NO: 39, 41, or 43.

If desired, for example, for diagnostic or assay purposes (e.g.,imaging), the chimeric antibody or antigen-binding portion thereof cancomprise a detectable label, e.g., as described herein for humanizedantibodies. All suitable methods and techniques described herein forhumanized antibodies of this invention can also be used for chimericantibodies of the invention.

In some embodiments, the anti-CCL20 antibody of the invention is a fullyhuman antibody. As used herein, the term “human antibody” means anyantibody in which the variable and constant domain sequences are humansequences. The term encompasses antibodies with sequences derived fromhuman genes, but which have been changed, e.g. to decrease possibleimmunogenicity, increase affinity, eliminate cysteines that might causeundesirable folding, etc. The term also encompasses such antibodiesproduced recombinantly in non-human cells, which might impartglycosylation not typical of human cells. Methods for preparing fullyhuman antibodies are known in the art. For example, human anti-CCL20antibodies may be identified through in vitro methods, such as phagedisplay, ribosome display (CAT), yeast display, and the like, or may beproduced from a human B-cell or a human hybridoma cell. Alternatively,human antibodies may be produced by immunizing with a CCL20 antigen anyof a number of non-human, transgenic animals comprising within theirgenomes some or all of human immunoglobulin heavy chain and light chainloci. In some embodiments, the non-human animal comprising humanimmunoglobulin genes is an animal that has a human immunoglobulin“minilocus” (e.g., GenPharm International, Inc.). In some embodiments,human anti-CCL20 antibodies are produced using a XENOMOUSE® (Abgenix,Inc., Fremont, Calif.), a HuMAb-Mouse® (Medarex, Inc.), a VelocImmune®mouse (Regeneron Pharmaaceuticals, Inc.), an AlivaMab Mouse (Ablexis,LLC), a KM™ mouse (Kirin Pharma USA, Inc.), or the like.

The present invention also relates to isolated and/or recombinantnucleic acids comprising sequences which encode a humanized antibody ora light chain or heavy chain thereof, mouse monoclonal antibody or alight chain or heavy chain thereof, chimeric antibody or a light chainor heavy chain thereof, or antigen-binding portions of any of the above,of the present invention. In some embodiments, the present inventionprovides a nucleic acid sequence selected from the group consisting ofSEQ ID NOS: 17-32, 51-56, 109, 111, and 113.

In some embodiments, the nucleic acid molecules of the invention includenucleic acid sequences that hybridize under highly stringent conditions,or that are at least 70%, 75%, 80%, 85%, 90%, 95%, 97%, 98% or 99%identical, to one or more of the nucleic acid sequences recited hereinor to a nucleic acid sequence encoding the amino acid sequence of any ofthe provided V_(H) or V_(L) sequences.

Nucleic acids referred to herein as “isolated” or “purified” are nucleicacids which have been separated away from the nucleic acids of thegenomic DNA or cellular RNA of their source of origin (e.g., as theyexist in cells or in a mixture of nucleic acids such as a library), andinclude nucleic acids obtained by methods described herein or othersuitable methods. In some embodiments, the isolated nucleic acids areessentially pure nucleic acids, nucleic acids produced by chemicalsynthesis, nucleic acids produced by combinations of biological andchemical methods, or recombinant nucleic acids which are isolated (see,e.g., Daugherty et al., Nucleic Acids Res. 19(9):2471-2476 (1991); Lewisand Crowe, Gene 101:297-302 (1991)).

A reference to a nucleotide sequence encompasses its complement unlessotherwise specified. Thus, a reference to a nucleic acid having aparticular sequence should be understood to encompass its complementarystrand, with its complementary sequence. The term “polynucleotide” asreferred to herein means a polymeric, possibly isolated, form ofnucleotides of at least 10 bases in length, either ribonucleotides ordeoxynucleotides or a modified form of either type of nucleotide. Theterm includes single and double stranded forms.

Nucleic acids referred to herein as “recombinant” are nucleic acidswhich have been produced by recombinant DNA methodology, including thosenucleic acids that are generated by procedures which rely upon a methodof artificial recombination, such as the polymerase chain reaction (PCR)and/or cloning into a vector using restriction enzymes. In someembodiments, the recombinant nucleic acids result from recombinationevents that occur through the natural mechanisms of cells, but areselected for after the introduction into the cells of nucleic acidsdesigned to allow and make probable a desired recombination event.

The present invention also relates more specifically to isolated and/orrecombinant nucleic acids comprising a nucleotide sequence which encodesa humanized antibody, mouse antibody or chimeric antibody, or anantigen-binding portion of said antibody, that has binding specificityfor human CCL20. In some embodiments, the antibody is a mouse antibodyof the present invention, a humanized antibody of the present inventionin which the nonhuman portion(s) are derived from a murine anti-CCL20monoclonal antibody; or a chimeric antibody of the present invention inwhich the nonhuman portion(s) are derived from a murine anti-CCL20monoclonal antibody.

In some embodiments, nucleic acids of the invention are used to producehumanized antibodies having binding specificity for human CCL20, mouseantibodies having binding specificity for human CCL20, and chimericantibodies having binding specificity for human CCL20. For example, anucleic acid (e.g., DNA (such as cDNA) or RNA) or one or more nucleicacids encoding a humanized antibody, mouse antibody or chimeric antibodyof the present invention can be incorporated into a suitable construct(e.g., a recombinant vector) for further manipulation of sequences orfor production of the encoded antibodies in suitable host cells.

Constructs or vectors suitable for the expression of a humanizedantibody having binding specificity for human CCL20, mouse antibodyhaving binding specificity for human CCL20 or chimeric antibody havingbinding specificity for human CCL20 are also provided. A variety ofvectors are available, including vectors which are maintained in singlecopy or multiple copies in a host cell, or which become integrated intothe chromosome(s) of a host cell. The constructs or vectors can beintroduced into a suitable host cell, and cells which express ahumanized antibody, mouse antibody or chimeric antibody of the presentinvention, can be produced and maintained in culture. A single vector ormultiple vectors can be used for the expression of a humanized antibody,mouse antibody or chimeric antibody having binding specificity for humanCCL20.

Suitable expression vectors, for example mammalian cell expressionvectors, can also contain a number of components, including, but notlimited to, one or more of the following: an origin of replication; aselectable marker gene; one or more expression control elements such asa transcriptional control element (e.g., a promoter, an enhancer, aterminator), one or more translation signals; and/or a signal sequenceor leader sequence (encoding a “signal peptide”) for membrane targetingor secretion. In a construct or vector, a signal sequence can beprovided by the construct or vector or other source. For example,transcriptional and/or translational signals can be used to directexpression.

In some embodiments, a promoter is provided for expression of anantibody or antibody chain of the invention in a suitable host cell. Insome embodiments, the promoter is constitutive. In some embodiments, thepromoter is inducible. The promoter may be operably linked to a nucleicacid encoding an antibody or antibody chain, or an antigen-bindingportion of said antibody or chain, such that it directs expression ofthe encoded polypeptide. A variety of suitable promoters for prokaryotic(e.g., lac, tac, T3, T7 promoters for E. coli) and eukaryotic (e.g.,yeast alcohol dehydrogenase (ADH1), SV40, CMV) hosts are available.Those of skill in the art will be able to select the appropriatepromoter for expressing an anti-CCL20 antibody or antigen-bindingportion thereof of the invention.

In some embodiments the vector encoding an antibody or antibody chain ofthe invention comprises a selectable marker for selection of host cellscarrying the vector. In some embodiments, the selectable marker is agene encoding a product that confers antibiotic or drug resistance thatmay be used in prokaryotic cells (e.g., β-lactamase gene (ampicillinresistance), Tet gene (tetracycline resistance), etc.) and eukaryoticcells (e.g., neomycin (G418 or geneticin), gpt (mycophenolic acid),ampicillin, or hygromycin resistance genes). In some embodiments, theselectable marker is dihydrofolate reductase, permitting selection withmethotrexate in a variety of hosts. In some embodiments, the selectablemarker is a gene encoding an auxotrophic marker of the host (e.g., LEU2,URA3, HIS3), e.g., for use in yeast. In some embodiments, the vector isa viral (e.g., baculovirus) or phage vector. In one embodiment, thevector is capable of integrating into the genome of the host cell (e.g.,retroviral vector). In some embodiments, the vector is a replicablevector and comprises an origin of replication.

The invention thus relates to isolated nucleic acid molecules thatencode the humanized antibody, humanized light chain, humanized heavychain, mouse antibody, mouse antibody light chain, mouse antibody heavychain, chimeric antibody, chimeric light chain, or chimeric heavy chainof this invention. The invention also relates to isolated nucleic acidmolecules that encode an antigen-binding portion of any of theseantibodies or their chains. Polypeptide sequences encoded by the nucleicacids of this invention are described above and in the followingExamples.

In some embodiments, a nucleic acid or vector of this invention encodesa heavy chain (or an antigen-binding portion thereof) or a light chain(or an antigen-binding portion thereof) of the invention. A host cellcontaining both the heavy chain-encoding nucleic acid and the lightchain-encoding nucleic acid, or nucleic acids encoding antigen-bindingportions of said heavy chain and said light chain, can be used to makean antibody comprising a heavy and a light chain (or an antigen-bindingportion of the antibody). The heavy chain-encoding nucleic acid and thelight chain-encoding nucleic acid can be placed on separate expressionvectors. They can also be placed on a single expression vector under thesame or different expression control. See, e.g., U.S. Pat. Nos.6,331,415 and 7,662,623.

Another aspect of the invention relates to a method of making ananti-human CCL20 antibody or an antigen-binding portion thereof of theinvention. The antibody or portion can be produced, for example, by theexpression of one or more recombinant nucleic acids encoding theantibody or portion in a suitable host cell. The host cell can beproduced using any suitable method. For example, one or more expressionconstructs described herein can be introduced into a suitable host cell,and the resulting cell can be maintained under conditions suitable forexpression of the construct(s) or vector(s). In some embodiments, theresulting cell is maintained in culture, in an animal, or in a plant.Suitable host cells can be prokaryotic, including bacterial cells suchas E. coli (e.g., strain DH5α™ (Invitrogen, Carlsbad, Calif.)), B.subtilis and/or other suitable bacteria; eukaryotic cells, such asfungal or yeast cells (e.g., Pichia pastoris, Aspergillus sp.,Saccharomyces cerevisiae, Schizosaccharomyces pombe, Neurospora crassa),or other lower eukaryotic cells, and cells of higher eukaryotes such asthose from insects (e.g., Drosophila Schneider S2 cells, Sf9 insectcells (WO 94/26087 (O'Connor), TN5B1-4 (HIGH 5) insect cells(Invitrogen), mammals (e.g., COS cells, such as COS-1 (ATCC AccessionNo. CRL-1650) and COS-7 (ATCC Accession No. CRL-1651), CHO (e.g., ATCCAccession No. CRL-9096), CHO DG44 (Urlaub and Chasin., Proc. Natl. Acac.Sci. USA 77(7):4216-4220 (1980)), 293 (ATCC Accession No. CRL-1573),HeLa (ATCC Accession No. CCL-2), CV1 (ATCC Accession No. CCL-70), WOP(Dailey et al., J. Virol. 54:739-749 (1985)), 3T3, 293T (Pear et al.,Proc. Natl. Acad. Sci. U.S.A. 90:8392-8396 (1993)), NS0 cells, SP2/0cells, HuT 78 cells and the like)), or plants (e.g., tobacco, lemna(duckweed), and algae). (See, for example, Ausubel et al., eds. CurrentProtocols in Molecular Biology, Greene Publishing Associates and JohnWiley & Sons Inc. (1993)). In some embodiments, the host cell is notpart of a multicellular organism (e.g., plant or animal). In certainembodiments, the host cell is an isolated host cell or is part of a cellculture.

The present invention also relates to cells comprising a nucleic acid ora vector of the invention. In some embodiments, the vector is anexpression vector. In some embodiments, one or more nucleic acidsencoding the heavy and light chains of a humanized antibody orantigen-binding portion thereof, the heavy and light chains of a mouseantibody or antigen-binding portion thereof, or the heavy and lightchains of a chimeric antibody or antigen-binding portion thereof, saidantibody or portion having binding specificity for human CCL20, or oneor more constructs comprising such nucleic acid(s), can be introducedinto a suitable host cell by a method appropriate to the host cellselected. In some embodiments, the method of introduction is, e.g.,transformation, transfection, electroporation, or infection. In someembodiments, the nucleic acid(s) are operably linked to one or moreexpression control elements. In certain embodiments, the nucleic acid(s)are in a vector, in a construct created by processes in the cell, orintegrated into the host cell genome. Host cells can be maintained underconditions suitable for expression. In some embodiments, theseconditions comprise the presence of an inducer, or of suitable media(supplemented with, e.g., appropriate salts, growth factors, antibiotic,nutritional supplements, etc.), whereby the encoded polypeptide(s) areproduced. These processes encompasses expression in a host cell (e.g., amammary gland cell) of a transgenic animal or plant (e.g., tobacco) (seee.g., WO 92/03918). In some embodiments, the antibodies or portions areisolated from the host cells, culture medium, or milk.

The invention also relates to fusion proteins in which an antibody orportion of the invention (e.g., a humanized antibody or portion) islinked to another moiety (e.g., a moiety which does not occur inantibodies as found in nature) in an N-terminal location, C-terminallocation or internal to the fusion protein. In some embodiments, thefusion protein can be produced by the insertion of a nucleic acidencoding an antibody sequence(s) into a suitable expression vector, suchas a pET vector (e.g., pET-15b, Novagen), a phage vector (e.g., pCANTAB5 E, Pharmacia), or other vector (e.g., pRIT2T Protein A fusion vector,Pharmacia). The resulting construct can be introduced into a suitablehost cell for expression. In some embodiments, the expressed fusionproteins are isolated or purified from a cell lysate by means of asuitable affinity matrix (see, e.g., Current Protocols in MolecularBiology (Ausubel et al., Eds., Vol. 2, Suppl. 26, pp. 16.4.1-16.7.8(1991))).

The invention relates to a host cell that comprises recombinant nucleicacid(s) encoding an antibody or portion, or heavy and/or light chainsthereof, provided herein (e.g., a humanized antibody, a humanized lightchain or a humanized heavy chain, a mouse antibody, a mouse light chainor a mouse heavy chain, a chimeric antibody, or a chimeric heavy chainor a chimeric light chain, of the invention).

The invention also relates to a host cell that comprises recombinantnucleic acid(s) encoding an antigen-binding portion of the antibody orits chains. In some embodiments, the host cell comprises a recombinantvector of the invention as referred to herein. In some embodiments, saidrecombinant vector is an expression vector. In certain embodiments, saidrecombinant vector is a mammalian cell expression vector.

The invention also relates to a method of preparing an antibody orportion, or a heavy or light chain thereof, of this invention. In oneembodiment, the method comprises maintaining a host cell of theinvention as described herein under conditions appropriate forexpression of the antibody or portion or heavy and/or light chainthereof. In some embodiments, the host cell contains one or moreisolated nucleic acids that encode the antibody or portion, or heavyand/or light chain thereof, of the invention. In some embodiments, thehost cell is cultured on a substrate or in suspension. In someembodiments, the method further comprises the step of purifying orisolating the antibody or antibody chain.

The invention further relates to a method of preparing antibodies orantigen-binding portions thereof through phage display. In someembodiments, a naïve antibody phage display library on CCL20 antigen ispanned. In some embodiments, a method of preparing antibodies throughguided selection is used (see, e.g., U.S. Patent Publication No. US2006-0251658 A1). In certain embodiments, a custom library built around,for example, a fixed heavy chain (and/or light chain) CDR3 region of aknown anti-CCL20 antibody is created. The CDR1 and CDR2 regions of theheavy and light chains can be derived from a naïve repertoire (Osburn etal., Methods 36:61-68 (2005)). In one embodiment, anti-CCL20 scFvs aregenerated from scFv naïve antibody libraries which are used to obtainmouse-human chimeric antibodies with the desired binding properties.These libraries may be screened for antibodies with the desired bindingproperties. In some embodiments, scFv phage libraries are used. Incertain embodiments, scFvs which recognize human CCL20 are isolated fromscFv guided selection libraries following a series of repeated selectioncycles on recombinant human CCL20 essentially as described in Vaughan etal. Nature Biotech. 14:309-314 (1996). In brief, following incubationwith the library, the immobilized antigen, which is pre-coupled toparamagnetic beads, and bound phage can be recovered by magneticseparation while unbound phage is washed away. Bound phage can then berescued as described by Vaughan et al. (1996; supra) and the selectionprocess repeated.

In a particular embodiment, a library is constructed consisting of theentire variable domain of the heavy chain of a mouse anti-CCL20 antibodyfused in a single chain format to a repertoire of naïve human lightchain variable regions. After selection, the human light chain variableregions that complement the mouse heavy chain variable regions areidentified. A library is then constructed consisting of the repertoireof human light chain variable regions selected as described above fusedin a single chain format to a chimeric heavy chain variable regionconsisting of naïve human CDR1 and CDR2 regions and a fixed CDR3 regionfrom the mouse anti-CCL20 antibody heavy chain variable domain. Afterselection for CCL20 binders, the best binding clones are selected. Fiveof the six CDR regions can be human in origin while the CDR3 of theheavy chain variable region can be identical to the original CDR3 of themouse heavy chain variable domain.

In some embodiments, selections are performed using CCL20 coupled toDYNABEADS M-270 amine (Dynal) according to the manufacturer'srecommendations. In some embodiments, selections using biotinylatedCCL20 can be prepared using the primary amine specific reagentsuccinimidyl-6-(biotinamido) hexanoate following the manufacturer'sinstructions (EZ link NHS LC Biotin, Pierce).

In some embodiments outputs from selections are tested as periplasmicpreparations in high throughput screens based on competition assayswhich measure the ability of the scFvs present in the periplasmicpreparation to compete for binding to CCL20.

Samples that are able to compete in the high throughput screens may besubjected to DNA sequencing as described in Vaughan et al. (1996; supra)and Osburn et al. (2005; supra). Clones may then be expressed andpurified as scFvs or IgGs and assessed for their ability to bind CCL20,neutralize CCL20 or a combination thereof, e.g., using assays such asantibody-dependent cell mediated cytotoxicity (ADCC) assay andcomplement dependent cytotoxicity (CDC) assay. Purified scFvpreparations can then be prepared as described in Example 3 of WO01/66754. Protein concentrations of purified scFv preparations weredetermined using the BCA method (Pierce). Similar approaches can be usedto screen for an optimal partner (the opposite chain) of a fixedfull-length antibody heavy or light chain or heavy or light chainvariable domain.

In another embodiment, an anti-human CCL20 antibody or anantigen-binding portion thereof, as described herein, is first used toselect heavy and light chain sequences having similar binding activitytoward CCL20, using the epitope imprinting methods described in PCTPublication No. WO 93/06213, incorporated herein by reference. Incertain embodiments, the antibody libraries used in this method are scFvlibraries prepared and screened as described in PCT Publication No. WO92/01047, McCafferty et al., Nature 348:552-554 (1990); and Griffiths etal., EMBO J. 12:725-734 (1993), all incorporated herein by reference. Incertain embodiments, the scFv antibody libraries are screened usinghuman CCL20 as the antigen.

Once initial V_(L) and V_(H) domains are selected, “mix and match”experiments can be performed, in which different pairs of the initiallyselected V_(L) and V_(H) segments are screened for CCL20 binding toselect preferred V_(L)/V_(H) pair combinations. Additionally, to furtherimprove the quality of the antibody,

the V_(L) and V_(H) segments of the preferred V_(L)/V_(H) pair(s) can berandomly mutated in a process analogous to the in vivo somatic mutationprocess responsible for affinity maturation of antibodies during anatural immune response. In some embodiments, the random mutations occurwithin the CDR3 region of V_(H) and/or V_(L). In certain embodiments,this in vitro affinity maturation is accomplished by, e.g., amplifyingV_(H) and V_(L) domains using PCR primers complimentary to the V_(H)CDR3 or V_(L) CDR3, respectively, wherein the primers have been “spiked”with a random mixture of the four nucleotide bases at certain positionssuch that the resultant PCR products encode V_(H) and V_(L) segmentsinto which random mutations have been introduced into the V_(H) and/orV_(L) CDR3 regions. These randomly mutated V_(H) and V_(L) segments canbe re-screened for binding to CCL20.

Following screening and isolation of an anti-CCL20 antibody of theinvention from a recombinant antibody display library, nucleic acidsencoding the selected antibody can be recovered from the display package(e.g., from the phage genome) and subcloned into other expressionvectors by standard recombinant DNA techniques. In some embodiments, thenucleic acid is further manipulated to create other antibody forms ofthe invention, as described herein. In certain embodiments, to express arecombinant human antibody isolated by screening of a combinatoriallibrary, the DNA encoding the antibody is cloned into a recombinantexpression vector and introduced into mammalian host cells, as describedherein.

In a particular embodiment, the invention provides a method of producinga hybridoma that secretes a monoclonal antibody that has bindingspecificity for human CCL20, comprising administering lymphocytes of aCCL20 transgenic mouse to a non-transgenic mouse having the same strain(e.g., CD1) as the human CCL20 transgenic mouse, thereby producing animmunized, non-transgenic mouse. Splenocytes of the immunized,non-transgenic mouse are contacted with immortalized cells, therebyproducing fused cells, and the fused cells are maintained underconditions in which hybridomas that secrete a monoclonal antibody havingbinding specificity for human CCL20 are produced, thereby producing ahybridoma that secretes a monoclonal antibody that has bindingspecificity for human CCL20.

The present invention also provides methods of preparing mutated formsof the anti-CCL20 antibodies and antigen-binding portions thereof of theinvention. In some embodiments, the antibodies or portions are mutatedin the variable domains of the heavy and/or light chains. In certainembodiments, said mutation alters one or more binding properties of theantibody or portion. In particular embodiments, a mutation is made inone or more of the CDR regions to increase or decrease the K_(D) of theanti-CCL20 antibody or portion, to increase or decrease k_(off), or toalter the binding specificity of the antibody or portion. Techniques insite-directed mutagenesis are well-known in the art. See e.g. Sambrooket al. and Ausubel et al., supra. In another embodiment, one or moremutations are made at an amino acid residue that is known to be changedcompared to the germline in a monoclonal antibody of the invention. Incertain embodiments, the mutations are made in a CDR region or frameworkregion of a variable domain, or in a constant domain. In a particularembodiment, the mutations are made in a variable domain. In someembodiments, one or more mutations are made at an amino acid residuethat is known to be changed compared to the germline in a CDR region orframework region of a variable domain of an antibody or portion of theinvention.

In another embodiment, the framework region is mutated so that theresulting framework region(s) have the amino acid sequence of thecorresponding germline gene. In certain embodiments, one or moremutations are made in a framework region or constant domain to increasethe half-life of the anti-CCL20 antibody or portion. See e.g. PCTPublication WO 00/09560. In certain embodiments, a mutation in aframework region or constant domain is made to alter the immunogenicityof the antibody, or to provide a site for covalent or non-covalentbinding to another molecule. According to the invention, a singleantibody or portion may have mutations in any one or more of the CDRs orframework regions of the variable domain or in the constant domain.

Anti-CCL20 antibodies or antigen-binding portions thereof of theinvention also can be produced transgenically through the generation ofa mammal or plant that is transgenic for the antibody heavy and lightchain sequences of interest and production of the antibody in arecoverable form therefrom. In some embodiments, the anti-CCL20antibodies or portions are produced in, and recovered from, the milk ofgoats, cows, or other mammals. See, e.g., U.S. Pat. Nos. 5,827,690,5,756,687, 5,750,172, and 5,741,957. In some embodiments, non-humantransgenic animals that comprise human antibody loci are immunized withhuman CCL20 or an immunogenic portion thereof, as described above.Methods for making antibodies in plants are described, e.g., in U.S.Pat. Nos. 6,046,037 and 5,959,177.

In some embodiments, non-human transgenic animals or plants are producedby introducing one or more nucleic acid molecules encoding an anti-CCL20antibody or portion of the invention into the animal or plant bystandard transgenic techniques. See Hogan and U.S. Pat. No. 6,417,429,supra. In certain embodiments, transgenic cells used for making thetransgenic animal can be embryonic stem cells or somatic cells or afertilized egg. In some embodiments, the non-human transgenic animals orplants are chimeric, nonchimeric heterozygotes, or nonchimerichomozygotes. See, e.g., Hogan et al., Manipulating the Mouse Embryo: ALaboratory Manual 2^(nd) ed., Cold Spring Harbor Press (1999); Jacksonet al., Mouse Genetics and Transgenics: A Practical Approach, OxfordUniversity Press (2000); and Pinkert, Transgenic Animal Technology: ALaboratory Handbook, Academic Press (1999). In some embodiments, thenon-human transgenic animals or plants have a targeted disruption andreplacement by a targeting construct that encodes a heavy chain and/or alight chain of interest. In certain embodiments, the transgenic animalsor plants comprise and express nucleic acid molecules encoding heavy andlight chains that specifically bind to human CCL20. The anti-CCL20antibodies or portions may be made in any non-human transgenic animal orplant. In particular embodiments, the non-human animals are mice, rats,sheep, pigs, goats, cattle or horses. The non-human transgenic animalmay express the encoded polypeptides in blood, milk, urine, saliva,tears, mucus and other bodily fluids.

The antibodies and antigen-binding portions thereof of the invention areuseful in neutralizing CCL20-induced chemoattraction of, e.g., CCR6+cells such as immature dendritic cells (DC), effector/memory T-cells,and B-cells. The antibodies and portions may thus be useful in treatinga variety of diseases and conditions such as inflammation, autoimmunediseases, and cancer. Examples of diseases and conditions that can betreated with the antibodies or antigen-binding portions of thisinvention include, without limitation, Grave's disease, vitiligo,hyperthyroidism, rheumatoid arthritis, psoriasis, atopic dermatitis,contact dermatitis, Crohn's disease, inflammatory bowel disease, B-cellmalignancies, breast adenocarcinoma, chronic hepatitis, contactdermatitis, glioblastoma, hepatocellular carcinoma, human papillomavirusinfection of the cervix, mycosis fungoides, pancreatic adenocarcinoma,periodontal disease, thyroid papillary carcinoma, pustulosis palmaris etplantaris, conditions associated with maculopapular exanthema,epidermolysis bullosa, alopecia greata, multiple sclerosis,polymyositis, dermatomyositis, Behcet's disease, acute generalizedexanthematous pustulosis, vasculitides, juvenile idiopathic arthritis,sarcoidosis, bronchial asthma, allergic rhinitis, renal allograftrejection, graft-versus-host disease, liver allograft rejection, chronicobstructive pulmonary disease, cystic fibrosis, glomerulonephritis,respiratory syncytial virus infection, multiple myeloma, and Langerhanscell histiocytosis. In some embodiments, the invention provides methodsfor treating any CCR6-associated condition.

Accordingly, the invention provides methods for treating inflammation,an autoimmune disease, or cancer by administering an effective amount ofan antibody or portion of the invention to a subject in need thereof. Insome embodiments, the subject is a human patient having or at risk forhaving an autoimmune disease, a cancer, and/or inflammation. In someembodiments, the antibody or portion is administered prophylactically toprevent onset or relapse of inflammation, autoimmune disease, or cancer.

Antibodies and antigen-binding portions thereof of this invention can beadministered to an individual (e.g., a human) alone or in conjunctionwith another agent in a combination therapy. The antibodies or portionscan be administered before, as an admixture with, separately butconcurrently with, or subsequent to administration of the additionalagent. In some embodiments, the additional agent is selected from thegroup including, but not limited to: inhibitors of immuno-stimulatorycytokines (e.g., anti-TNF-α MAbs), immune cell eliminators (e.g.,anti-CD20 MAbs), blockers of accessory molecules (e.g. Abatacept),disease-modifying agents for rheumatic diseases (e.g., non-steroidalanti-inflammatory drugs (NSAIDs), methotrexate, retinoic acids, andvitamin D3 analogs), and immunosuppressants (e.g., calcineurininhibitors). In some embodiments, the additional agent is selected fromthe group including, but not limited to: steroidal agents,immunomodulatory agents, somatostatic agents, conventionalimmunotherapeutic agents, cytokines or cytokine antagonists, and/orgrowth factors or growth factor antagonists. In some embodiments,anti-CCL20 antibody therapy can be used in conjunction withstandard-of-care cancer treatment such as chemotherapy, surgery, orradiation, or with another targeted therapy such as anti-VEGF antibodytherapy. In some embodiments, anti-CCL20 antibody therapy can be used inconjunction with a prophylactic regimen. In one embodiment, a syntheticpeptide mimetic can be administered in conjunction with an antibody ofthe present invention. In another embodiment, hormone therapy can beadministered in conjunction with an antibody or portion of the presentinvention.

In one embodiment, the antibodies or antigen-binding portions of theinvention are administered alone or in combination with ananti-inflammatory agent. Anti-inflammatory agents that may beadministered with the antibodies of the invention include, but are notlimited to, corticosteroids (e.g. betamethasone, budesonide, cortisone,dexamethasone, hydrocortisone, methylprednisolone, prednisolone,prednisone, and triamcinolone), nonsteroidal anti-inflammatory drugs(e.g., balsalazide, celecoxib, diclofenac, diflunisal, etodolac,fenoprofen, floctafenine, flurbiprofen, ibuprofen, indomethacin,ketoprofen, meclofenamate, mefenamic acid, meloxicam, nabumetone,naproxen, olsalazine, oxaprozin, phenylbutazone, piroxicam, salsalate,sulindac, tenoxicam, tiaprofenic acid, and tolmetin), as well asacetaminophen, antihistamines, aminoarylcarboxylic acid derivatives,arylacetic acid derivatives, arylbutyric acid derivatives,arylcarboxylic acids, arylpropionic acid derivatives, pyrazoles,pyrazolones, salicylic acid derivatives (e.g. sulfasalazone andmesalamine), thiazinecarboxamides, e-acetamidocaproic acid,S-adenosylmethionine, 3-amino-4-hydroxybutyric acid, amixetrine,bendazac, benzydamine, bucolome, difenpiramide, ditazol, emorfazone,guaiazulene, nabumetone, nimesulide, orgotein, oxaceprol, paranyline,perisoxal, pifoxime, proquazone, proxazole, and tenidap.

Conventional nonspecific immunosuppressive agents that may beadministered in combination with the antibodies of the inventioninclude, but are not limited to, steroids, cyclosporine, cyclosporineanalogs, cyclophosphamide methylprednisone, prednisone, azathioprine,FK-506, 15-deoxyspergualin, natalizumab, and other immunosuppressiveagents that act by suppressing the function of responding T cells.

In some embodiments, the antibodies or antigen-binding portions of theinvention are administered in combination with immunosuppressants.Immunosuppressant preparations that may be administered with theantibodies of the invention include, but are not limited to, ORTHOCLONE™(OKT3), SANDIMMUNE™/NEORAL™/SANGDYA™ (cyclosporin), PROGRAF™(tacrolimus), CELLCEPT™ (mycophenolate), Azathioprine,glucorticosteroids, AVONEX™ (interferon-beta 1A), and RAPAMUNE™(sirolimus). In one embodiment, immunosuppressants may be used toprevent rejection of organ or bone marrow transplantation.

In some embodiments, the antibodies or antigen-binding portions of theinvention are administered in combination with a chemotherapeutic agent.Chemotherapeutic agents that may be administered with the antibodies ofthe invention include, but are not limited to, antibiotic derivatives(e.g., doxorubicin, bleomycin, daunorubicin, and dactinomycin);antiestrogens (e.g., tamoxifen); antimetabolites (e.g., fluorouracil,5-FU, floxuridine, interferon alpha-2b, glutamic acid, plicamycin,mercaptopurine, and 6-thioguanine); cytotoxic agents (e.g., carmustine,BCNU, lomustine, CCNU, cytosine arabinoside, cyclophosphamide,estramustine, hydroxyurea, procarbazine, mitomycin, busulfan,cis-platin, and vincristine sulfate); hormones (e.g.,medroxyprogesterone, estramustine phosphate sodium, ethinyl estradiol,estradiol, epinephrine, megestrol acetate, methyltestosterone,diethylstilbestrol diphosphate, chlorotrianisene, and testolactone);nitrogen mustard derivatives (e.g., mephalen, chorambucil,mechlorethamine (nitrogen mustard) and thiotepa); steroids andcombinations (e.g., bethamethasone sodium phosphate); and others (e.g.,dicarbazine, asparaginase, mitotane, vincristine sulfate, vinblastinesulfate, rituximab, and etoposide).

In some embodiments, the antibodies or antigen-binding portions of theinvention are administered in combination with a TNF antagonist. TNFantagonists that may be administered with the antibodies orantigen-binding portions of the invention include, but are not limitedto, infliximab (REMICADE™), adalimumab (HUMIRA™), certolizumab pegol(CIMZIA™), golimumab (SIMPONI™), etanercept (ENBREL™), xanthinederiviatives (e.g. pentoxifyline) and bupropion (WELLBURTIN™, ZYBAN™)

In some embodiments, the antibodies or antigen-binding portions of theinvention are administered alone or in combination with one or moreintravenous immune globulin preparations. Intravenous immune globulinpreparations that may be administered with the antibodies or portions ofthe invention include, but not limited to, GAMMAR™, IVEEGAM™,SANDOGLOBULIN™ GAMMAGARD S/D™, and GAMIMUNE™. In some embodiments, theantibodies or antigen-binding portions of the invention are administeredin combination with intravenous immune globulin preparations intransplantation therapy (e.g., bone marrow transplant).

In some embodiments, the antibodies or antigen-binding portions of theinvention are administered in combination with cytokines or cytokineantagonists. In some embodiments, the antibodies or antigen-bindingportions of the invention may be administered with any cytokine orcytokine antagonist, including, but not limited to, IL2, IL3, IL4, IL5,IL6, IL7, IL10, IL12, IL13, IL15, anti-CD40, CD40L, IFN-γ and TNF-α orany antagonist thereof. In some embodiments, the antibodies orantigen-binding portions of the invention may be administered with anyinterleukin or interleukin antagonist, including, but not limited to,IL-1alpha, IL-1beta, IL-2, IL-3, IL-4, IL-5, IL-6, IL-7, IL-8, IL-9,IL-10, IL-11, IL-12, IL-13, IL-14, IL-15, IL-16, IL-17, IL-18, IL-19,IL-20, and IL-21 or any antagonist thereof.

In some embodiments, the antibodies or antigen-binding portions of theinvention are administered in combination with one or more chemokines orchemokine antagonists. In specific embodiments, the antibodies orantigen-binding portions of the invention are administered incombination with an α(C×C) chemokine selected from the group consistingof gamma-interferon inducible protein-10 (γIP-10), interleukin-8 (IL-8),platelet factor-4 (PF4), neutrophil activating protein (NAP-2), GRO-α,GRO-β, GRO-γ, neutrophil-activating peptide (ENA-78), granulocytechemoattractant protein-2 (GCP-2), and stromal cell-derived factor-1(SDF-1, or pre-B cell stimulatory factor (PBSF)); and/or a β (CC)chemokine selected from the group consisting of: RANTES (regulated onactivation, normal T expressed and secreted), macrophage inflammatoryprotein-1 alpha (MIP-1α), macrophage inflammatory protein-1 beta(MIP-1β), monocyte chemotactic protein-1 (MCP-1), monocyte chemotacticprotein-2 (MCP-2), monocyte chemotactic protein-3 (MCP-3), monocytechemotactic protein-4 (MCP-4) macrophage inflammatory protein-1 gamma(MIP-1γ), macrophage inflammatory protein-3 alpha (MIP-3α), macrophageinflammatory protein-3 beta (MIP-3β), macrophage inflammatory protein-4(MIP-4/DC-CK-1/PARC), eotaxin, Exodus, and 1-309; and/or the γ(C)chemokine, lymphotactin; or any antagonist thereof.

In some embodiments, the antibodies or antigen-binding portions of theinvention are administered with chemokine beta-8, chemokine beta-1,and/or macrophage inflammatory protein-4, or any antagonist thereof. Ina preferred embodiment, the antibodies or antigen-binding portions ofthe invention are administered with chemokine beta-8 or an antagonistthereof.

In some embodiments, the antibodies or antigen-binding portions of theinvention are administered in combination with an IL-4 antagonist. IL-4antagonists that may be administered with the antibody and antibodycompositions of the invention include, but are not limited to: solubleIL-4 receptor polypeptides, multimeric forms of soluble IL-4 receptorpolypeptides, anti-IL-4 receptor antibodies that bind the IL-4 receptorwithout transducing the biological signal elicited by IL-4, anti-IL4antibodies that block binding of IL-4 to one or more IL-4 receptors, andmuteins of IL-4 that bind IL-4 receptors but do not transduce thebiological signal elicited by IL-4. Preferably, the anti-IL4 antibodiesemployed according to this method are monoclonal antibodies;antigen-binding portions thereof may also be employed.

In some embodiments, the antibodies or antigen-binding portions of theinvention are administered in combination with members of the TNFfamily, or antagonists thereof. In some embodiments, the antibodies orantigen-binding portions of the invention are administered incombination with agents that include, but are not limited to, solubleforms of TNF-α, lymphotoxin-alpha (LT-alpha, also known as TNF-beta),LT-beta (found in complex heterotrimer LT-alpha2-beta), OPGL, FasL,CD27L, CD30L, CD40L, 4-1BBL, DcR3, OX40L, TNF-gamma (InternationalPublication No. WO 96/14328), AIM-I (International Publication No. WO97/33899), endokine-alpha (International Publication No. WO 98/07880),OPG, neutrokine-alpha (International Publication No. WO 98/18921), OX40,nerve growth factor (NGF), soluble forms of Fas, CD30, CD27, CD40 4-IBB,TR2 (International Publication No. WO 96/34095), DR3 (InternationalPublication No. WO 97/33904), DR4 (International Publication No. WO98/32856), TR5 (International Publication No. WO 98/30693), TR6(International Publication No. WO 98/30694), TR7 (InternationalPublication No. WO 98/41629), TRANK, TR9 (International Publication No.WO 98/56892), TR10 (International Publication No. WO 98/54202), 312C2(International Publication No. WO 98/06842), TR12, and soluble formsCD154, CD70, and CD153; or any antagonist thereof.

As used herein, the term “therapeutically effective amount” refers to anamount of the administered therapeutic agent that will relieve orprevent to some extent one or more of the symptoms of the disorder beingtreated. An effective amount of anti-CCL20 antibody or antigen-bindingportion thereof for treating a disease is an amount that helps thetreated subject to reach one or more desired clinical end points.

In some embodiments, to minimize immunogenicity, a humanized antibody orportion is used to treat a human patient in therapeutic methods andcompositions of this invention. In cases where repeated administrationis not necessary, in some embodiments, it may also be appropriate toadminister a mouse or chimeric antibody or portion of the invention to ahuman patient.

In some embodiments, the antibodies or antigen-binding portions thereofof the invention may be used to treat an individual who has previouslybeen treated with, e.g., inhibitors of immuno-stimulatory cytokines(e.g., anti-TNF-α MAbs), immune cell eliminators (e.g., anti-CD20 MAbs),and/or blockers of accessory molecules (e.g. Abatacept). In someembodiments, the antibodies or portions are used to treat a patient whohas developed primary non-responsiveness or a gradual decline inresponse rate to these other treatments.

The antibody or antigen-binding portion thereof of this invention can beadministered in a single unit dose or multiple doses at any time pointdeemed appropriate by a health care provider. The dosage can bedetermined by methods known in the art and can be dependent, forexample, upon the individual's age, sensitivity, tolerance and overallwell-being. Any administration method accepted in the art may beemployed suitably for the antibodies and portions of the invention,including, but not necessarily limited to, parenteral (e.g.,intravenous, intraarterial, intramuscular, intrathecal, intraperitoneal,subcutaneous injection), oral (e.g., dietary), locally, topical,inhalation (e.g., intrabronchial, intranasal or oral inhalation,intranasal drops), or rectal, depending on the disease or condition tobe treated. In one embodiment, the antibody or portion is administeredparenterally.

Formulation will vary according to the route of administration selected(e.g., solution, emulsion). An appropriate composition comprising theantibody or portion to be administered can be prepared in aphysiologically acceptable vehicle or carrier. The composition cancomprise multiple doses or be a single unit dose composition. Forsolutions or emulsions, suitable carriers include, for example, aqueousor alcoholic/aqueous solutions, emulsions, or suspensions, includingsaline and buffered media. Parenteral vehicles can include sodiumchloride solution, Ringer's dextrose, dextrose and sodium chloride,lactated Ringer's or fixed oils. Intravenous vehicles can includevarious additives, preservatives, or fluid, nutrient or electrolytereplenishers (See, generally, Remington's Pharmaceutical Sciences, 17thEdition, Mack Publishing Co., PA, 1985). For inhalation, the compoundcan be solubilized and loaded into a suitable dispenser foradministration (e.g., an atomizer, nebulizer or pressurized aerosoldispenser).

Dosage regimens may be adjusted to provide the optimum desired response.In certain embodiments, a single bolus may be administered, severaldivided doses may be administered over time, or the dose may beproportionally reduced or increased as indicated by the exigencies ofthe therapeutic situation. It is especially advantageous to formulateparenteral compositions in dosage unit form for ease of administrationand uniformity of dosage. Dosage unit form, as used herein, refers tophysically discrete units suited as unitary dosages for thepatients/subjects to be treated; each unit containing a predeterminedquantity of active compound calculated to produce the desiredtherapeutic effect in association with the required pharmaceuticalcarrier. The specification for the dosage unit forms of the inventionare generally dictated by and directly dependent on (a) the uniquecharacteristics of the therapeutic agent and the particular therapeuticor prophylactic effect to be achieved, and (b) the limitations inherentin the art of compounding such an active compound for the treatment ofsensitivity in individuals.

Thus, the skilled artisan would appreciate, based upon the disclosureprovided herein, that the dose and dosing regimen is adjusted inaccordance with methods well-known in the therapeutic arts. That is, themaximum tolerable dose can be readily established, and the effectiveamount providing a detectable therapeutic benefit to a patient may alsobe determined, as can the temporal requirements for administering eachagent to provide a detectable therapeutic benefit to the patient.Accordingly, while certain dose and administration regimens areexemplified herein, these examples in no way limit the dose andadministration regimen that may be provided to a patient in practicingthe present invention.

It is to be noted that dosage values may vary with the type and severityof the condition to be alleviated, and may include single or multipledoses. It is to be further understood that for any particular subject,specific dosage regimens should be adjusted over time according to theindividual need and the professional judgment of the personadministering or supervising the administration of the compositions, andthat dosage ranges set forth herein are exemplary only and are notintended to limit the scope or practice of the claimed composition.Further, the dosage regimen with the compositions of this invention maybe based on a variety of factors, including the type of disease, theage, weight, sex, medical condition of the patient, the severity of thecondition, the route of administration, and the particular antibodyemployed. Thus, the dosage regimen can vary widely, but can bedetermined routinely using standard methods. For example, doses may beadjusted based on pharmacokinetic or pharmacodynamic parameters, whichmay include clinical effects such as toxic effects and/or laboratoryvalues. The present invention thus encompasses intra-patientdose-escalation as determined by the skilled artisan. Methods ofdetermining appropriate dosages and regimens are well-known in therelevant art and would be understood to be encompassed by the skilledartisan once provided with the teachings disclosed herein.

In some embodiments, for administration to human subjects, the totalmonthly dose of the antibodies or antibody portion of the invention isin the range 0.5-1200 mg per patient, depending, of course, on the modeof administration. In certain embodiments, an intravenous monthly doserequires about 1-1000 mg/patient. The total monthly dose may beadministered in single or divided doses and may, at the physician'sdiscretion, fall outside of the typical range given herein.

In certain embodiments, a range for a therapeutically orprophylactically effective amount of an antibody or antibody portion ofthe invention is 1-1000 mg/kg/patient/month. In one embodiment, theantibody or portion thereof of the invention may be administered atabout 1-200 or 1-150 mg/patient/month. In certain embodiments, theantibody or portion is administered in a 1, 2, 3, 4, 5, 6, 7, 8, 9, or10 mg/kg/patient shot 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 times a month. Ina particular embodiment, the antibody or portion is administered in a 1to 5 mg/kg/patient shot 1 or 2 times a month.

The antibodies and antigen-binding portions thereof of the presentinvention also are useful in a variety of processes with applications inresearch and diagnosis. In some embodiments, the antibodies and portionsare used to detect, isolate, and/or purify human CCL20 or variantsthereof (e.g., by affinity purification or other suitable methods suchas flow cytometry, e.g., for cells, such as lymphocytes, in suspension),and to study human CCL20 structure (e.g., conformation) and function.For in vitro applications, wherein immunogenicity of the antibody is nota concern, the mouse and chimeric antibodies and antigen-bindingprotions thereof of this invention will be useful in addition tohumanized antibodies.

The antibodies or antigen-binding portions thereof of the presentinvention can be used in diagnostic applications (e.g., in vitro, exvivo). In some embodiments, the humanized antibodies or portions of thepresent invention are used to detect and/or measure the level of humanCCL20 in a sample. In certain embodiments, the sample comprises, e.g.,cells or tissues expressing human CCL20, and/or body fluids such as aninflammatory exudate, blood, serum, and/or bowel fluid bearing humanCCL20. A sample can be obtained from an individual and an antibody orportion described herein can be used in a suitable immunological methodto detect and/or measure human CCL20 expression, including methods suchas flow cytometry (e.g., for cells in suspension such as lymphocytes),enzyme-linked immunosorbent assays (ELISA), including chemiluminescenceassays, radioimmunoassay, and immunohistology.

In one embodiment, a method of detecting human CCL20 in a samplecomprises contacting the sample with an antibody or portion of thepresent invention under conditions suitable for specific binding of theantibody or portion to human CCL20 and detecting antibody-CCL20complexes which are formed. In one embodiment, the antibodies orportions described herein can be used to analyze normal versus inflamedtissues (e.g., from a human) for human CCL20 reactivity and/orexpression (e.g., immunohistologically) to detect associations betweenincreased expression of human CCL20 (e.g., in affected tissues) and oneor more disorders selected from, but not limited to, Grave's disease,vitiligo, hyperthyroidism, rheumatoid arthritis, psoriasis, atopicdermatitis, contact dermatitis, Crohn's disease, inflammatory boweldisease, B-cell malignancies, breast adenocarcinoma, chronic hepatitis,contact dermatitis, glioblastoma, hepatocellular carcinoma, humanpapillomavirus infection of the cervix, mycosis fungoides, pancreaticadenocarcinoma, periodontal disease, thyroid papillary carcinoma,pustulosis palmaris et plantaris, conditions associated withmaculopapular exanthema, epidermolysis bullosa, alopecia greata,multiple sclerosis, polymyositis, dermatomyositis, Behcet's disease,acute generalized exanthematous pustulosis, vasculitides, juvenileidiopathic arthritis, sarcoidosis, bronchial asthma, allergic rhinitis,renal allograft rejection, graft-versus-host disease, liver allograftrejection, chronic obstructive pulmonary disease, cystic fibrosis,glomerulonephritis, respiratory syncytial virus infection, multiplemyeloma, Langerhans cell histiocytosis, or other conditions. Thus, theantibodies of the present invention permit immunological methods ofassessment of the presence of human CCL20 in normal and inflamedtissues, through which the presence of disease, disease progress and/orthe efficacy of anti-human CCL20 therapy in the treatment of disease,e.g., inflammatory and/or immune disease, can be assessed.

The methods and techniques of the present invention are generallyperformed according to conventional methods well known in the art and asdescribed in various general and more specific references that are citedand discussed throughout the present specification unless otherwiseindicated. See, e.g., Sambrook J. & Russell D. Molecular Cloning: ALaboratory Manual, 3rd ed., Cold Spring Harbor Laboratory Press, ColdSpring Harbor, N.Y. (2000); Ausubel et al., Short Protocols in MolecularBiology: A Compendium of Methods from Current Protocols in MolecularBiology, Wiley, John & Sons, Inc. (2002); Harlow and Lane UsingAntibodies: A Laboratory Manual, Cold Spring Harbor Laboratory Press,Cold Spring Harbor, N.Y. (1998); and Coligan et al., Short Protocols inProtein Science, Wiley, John & Sons, Inc. (2003). Enzymatic reactionsand purification techniques are performed according to manufacturer'sspecifications, as commonly accomplished in the art or as describedherein. The nomenclature used in connection with, and the laboratoryprocedures and techniques of, analytical chemistry, synthetic organicchemistry, and medicinal and pharmaceutical chemistry described hereinare those well known and commonly used in the art.

Unless otherwise defined, all technical and scientific terms used hereinhave the same meaning as commonly understood by one of ordinary skill inthe art to which this invention belongs. Exemplary methods and materialsare described below, although methods and materials similar orequivalent to those described herein can also be used in the practice ortesting of the present invention. All publications and other referencesmentioned herein are incorporated by reference in their entirety. Incase of conflict, the present specification, including definitions, willcontrol. Although a number of documents are cited herein, this citationdoes not constitute an admission that any of these documents forms partof the common general knowledge in the art. Throughout thisspecification and claims, the word “comprise,” or variations such as“comprises” or “comprising” will be understood to imply the inclusion ofa stated integer or group of integers but not the exclusion of any otherinteger or group of integers. The materials, methods, and examples areillustrative only and not intended to be limiting.

In order that this invention may be better understood, the followingexamples are set forth. These examples are for purposes of illustrationonly and are not to be construed as limiting the scope of the inventionin any manner.

Example 1 Involvement of CCL20 in Autoimmune/Inflammatory Disorders

To obtain evidence for the involvement of CCL20 in autoimmune andinflammatory conditions, we studied the effects of knocking out theCCL20 receptor CCR6 in a type II collagen-induced arthritis (CIA) mouserheumatoid arthritis (RA) model. CCR6 wild type mice, CCR6^(+/−)(heterozygous) mice, and CCR6^(−/−) (homozygous) knock-out mice (eachn=10) were immunized at the base of the tail with bovine type IIcollagen (150 μg/mouse) emulsified in complete Freund's adjuvant(Chondrex, #7001). Three weeks later, a booster injection of the sameamount of bovine type II collagen emulsion in incomplete adjuvant wasadministered at the base of the tail. We graded the severity ofarthritic symptoms in the paws of each mouse as described in Griswold etal., Arthritis & Rheumatism 31(11):1406-1412 (1988). Briefly, we gradedthe articular lesions of the extremities distal to the elbow or knee ona scale of 0 to 4 based on the number of joints involved and the degreeof erythema and swelling. Arthritis scores were calculated as the sum ofthe scores of all four paws for each animal. CCR6 wild type animalsdeveloped arthritis, while CCR6^(−/−) animals showed strong resistanceto the disease (FIG. 1). Interestingly, CCR6^(+/−) animals alsoexhibited some levels of resistance, indicating that total abrogation ofCCL20 function is not required to counteract the arthritic phenotype.

In addition, we immunohistochemically analyzed the hind paws of mice forthe presence of CD3-positive T-cells or F4/80-positive macrophages. Onday 43, mice were harvested and their hind paws were fixed in formalin.The paws were then sectioned and placed on slides. After decalcificationof the paw samples, the slides were stained immunohistochemically withanti-CD3 (#N1580, DAKO) and F4/80 antibodies (Clone CI:A3-1, AbDSerotec). The intensity of staining was scored by two independentobservers by scanning the samples with an Aperio instrument (AperioTechnologies). The scoring scale was as follows: 0, normal; 1, lightstaining throughout the paw or intense staining in one digit; 2,moderate staining in multiple digits; 3, intense staining in multipledigits or moderate throughout; 4, intense staining throughout all digitsand paw. The number of T-cells (FIG. 2) and macrophages (FIG. 3)infiltrating the CIA-induced lesions were strongly reduced in bothCCR6^(+/−) and CCR6^(−/−) mice.

We also studied the effects of knocking out CCR6 in adinitrofluorobenzene (DNFB)-induced allergic contact dermatitis mousemodel. Two groups of six mice each (one group of mice wild-type for CCR6and one group CCR6 deficient) were sensitized by brushing 25 μl of 0.4%DNFB solution (in 4:1 acetone:olive oil) on the shaved abdomen for twosuccessive days (days 0 and 1). On day 5, mice were re-challenged byapplying 20 μl of 0.1% DNFB (in 4:1 acetone:olive oil) to one side ofone ear. As an indicator of edema, ear thickness was measured beforeDNFB challenge and at 24 hr after the last challenge on Day 5 by using athickness gauge. CCR6 deficient mice demonstrated almost no increasedear thickness after treatment with DNFB, indicating resistance againstDNFB-induced contact hypersensitivity (FIG. 4).

These data support a role for CCR6 and CCL20 in autoimmune/inflammatorydisorders.

Example 2 Inhibition of CCL20-Induced Chemotaxis by Hamster Anti-MouseCCL20 2F5-5 MAb

To further explore the role of CCL20 as a potential therapeutic target,we performed experiments using a hamster anti-mouse CCL20 antibody(2F5-5). We first characterized the ability of the 2F5-5 MAb to bind tomouse, human, and rhesus CCL20. Soluble CCL20-secreted alkalinephosphatase (SEAP) antigens of each species were prepared as follows.The cDNAs encoding the CCL20s of human, cynomolgus monkey, rhesusmonkey, rat, and mouse were amplified and subcloned into a pcDNA3.1 (+)dSalI SEAP vector, which contains SEAP cDNA and has its SalI sitedeleted (pcDNA 3.1 (+) purchased from Invitrogen; SEAP cDNA derived froma pSEAP-Enhancer vector, Clontech). The expression vectors weretransfected into the human embryo kidney cell line HEK293EBNA (HEK293E,Invitrogen). The HEK293E cells were inoculated with DMEM (Invitrogen)supplemented with 10% fetal bovine serum on the day before transfection.On the day of transfection, the culture medium was replaced withOPTI-MEM II serum free media (Invitrogen). The expression vectors weretransfected using TransIT LT1 (TAKARA Bio Inc., Shiga, Japan) accordingto the manufacturer's protocol. After 3 days of incubation at 5% CO₂ and37° C., culture supernatants were harvested. The concentration ofCCL20-SEAP in each culture was measured by using the Great EscAPe SEAPChemiluminescence Kit 2.0 (Clontech).

We then performed surface plasmon resonance (Biacore™) binding studiesto measure the binding of the 2F5-5 MAb to mouse, human, rhesus, andcynomolgus CCL20-SEAP antigens. Anti-SEAP (monoclonal anti-mouseplacental alkaline phosphatase, Thermo Scientific, Cat #MAI-19354, Lot#KL12748M) was immobilized on a CM5 sensor chip (GE Healthcare) using astandard NHS/EDC amine coupling procedure. SEAP-tagged mouse CCL20 (100nM in supernatant, ID735, Lot #091130) was diluted to 5 nM with TBS-Pcontaining 0.005% Tween-20 and captured on the CM5 sensor chip. 0.2 nMto 80 nM dilutions of hamster anti-mouse CCL20 (2F5-5, Lot #060215, 2mg/ml) in TBS-P were injected over the sensor chip at a flow rate of 30μl/min. Association and dissociation of hamster anti-mouse CCL20 withmouse CCL20 were monitored for 4 min and 16 min, respectively. The chipsurface was regenerated between injections using 10 mM glycine, pH 2.25.Sensograms were double referenced by subtracting hamster anti-mouseCCL20 injections into a reference cell without captured mouse CCL20 anda TBS-P injection over captured mouse CCL20. The sensograms wereanalyzed using a 1:1 Langmuir binding model in BIAevaluation software(GE Healthcare, version 3.2). In this assay, 2F5-5 MAb binds to mouseCCL20 with an affinity of 32 μM, but does not bind to human, rhesus, orcynomolgus CCL20 (Table 1).

TABLE 1 2F5-5 MAb binding affinity to CCL20 orthologs k_(a) k_(d) K_(D)CCL20 Ortholog (×10⁵ M⁻¹sec⁻¹) (×10⁻⁵ sec⁻¹) (×10⁻¹¹ M) Mouse 9.63 0.4740.492 Human Not detectable Not detectable Not detectable Rhesus Notdetectable Not detectable Not detectable Cynomolgus Not detectable Notdetectable Not detectable

To test the effect of 2F5-5 MAb on CCL20 function, we evaluated itsneutralizing activity against mouse CCL20 using an in vitro chemotaxisassay. CCR6-transduced B300.19 cells and recombinant mouse CCL20 (1 nM)were added to transwell culture plates. Four hours later, the cells thatmigrated into the lower chamber were counted by fluorescence-activatedcell sorting (FACS). 2F5-5 MAb completely inhibited chemotaxis at 1μg/ml with an estimated IC₅₀ value of 0.04 μg/ml (0.27 nM) (FIG. 5). Bycontrast, a control hamster antibody (10 μg/ml) did not inhibitchemotaxis (100% control; data not shown).

Example 3 Humanization of Mouse Anti-CCL20 MAbs

To obtain monoclonal antibodies against human CCL20, we generated apanel of mouse anti-human CCL20 antibodies. Recombinant human CCL20(R&D, #360-MP-025/CF, 17.5 μg/head) emulsified with Freund's completeadjuvant (Mitsubishi-Kagaku Yatron, RM606-1) was injected subcutaneouslyinto the footpads of the mice. Two consecutive injections were thenadministered every three days. Three days after the final immunization,the animals were sacrificed and the inguinal lymph-node cells were fusedwith P3U1 myeloma cells at a 2:1 to 10:1 ratio in the presence of 50%polyethylene glycol. The cells were then cultured in 96-well plasticplates.

Sandwich ELISA was used for primary screening. A 96-well plate wascoated with a polyclonal anti-human IgG antibody (Jackson, #709-005-149,2 μg/ml in PBS (−)). After overnight incubation at 4° C., the wells wereblocked with 1×Block-Ace (Dainippon Sumitomo Pharma, UK-B 80) for 1 hourat room temperature. The wells were then washed with 0.02% Tween 20/PBS(−), after which 1 nM human CCL20-hIgG Fc chimera protein in 0.02% Tween20/PBS (−) was added to the wells (50 μl/well). After a one hourincubation at room temperature, three additional washes were performedas described above. Culture supernatants from each hybridoma were thendiluted twofold with 20% FBS and 200 μg/ml human IgG (MitsubishiWelpharma) in 0.02% Tween 20/PBS (−), added to the wells, and incubatedfor one hour at room temperature. After three further washes, the wellswere incubated with horseradish peroxidase-conjugated anti-mouse IgGantibody (Jackson, #715-035-150, 5000 fold dilution with 0.02% Tween20/PBS (−)) for one hour at room temperature. The wells were then washedthree times and incubated in a TMBZ (3,3′,5,5′-tetramethylbenzidine)solution for 15-30 minutes. An equal volume of 2 M H₂SO₄ was then addedto stop the reaction, and the optical density was read at 450 nm by ARVO(PerkinElmer). The sandwich ELISA identified 24 positive wells.

We then performed a chemotaxis assay as a secondary screen. Thechemotaxis assay was performed in transwell culture plates (MultiScreenpore 5 μm, Millipore, #MAMIC 5S10). First, 50 μl/well of 300 ng/mlrecombinant human CCL20 (R&D, #360-MP-025/CF) in chemotaxis buffer (0.5%BSA, 0.5% FBS, 20 mM HEPES (pH 7.4), 50 μM 2-mercaptoethanol in RPMI1640(Invitrogen)) was pre-incubated with 100 μl/well of culture supernatantsfrom the hybridomas at room temperature for 30 minutes (for a finalCCL20 concentration of 100 ng/ml) in the lower wells of the plates.After 30 minutes, B300.19 cells transfected with human CCR6 (SEQ ID NO:104) (2×10⁵ cells/75 μl) were applied to the upper wells and incubatedin a 5% CO₂ incubator at 37° C. for 4 hours. Following the incubation,150 μl from the lower wells were harvested and fixed with 50 μl of 4%PFA/PBS (−). 30 μl of each sample were applied to the FACSCantoII cellanalyzer (BD Biosciences) to count migrated cells. Neutralizing activitywas found in four wells.

We then used standard limiting dilution to obtain hybridoma clones fromthe four positive wells. We confirmed the neutralizing activity ofsupernatant from each clone using the in vitro chemotaxis assay. Threeof the mouse anti-human CCL20 monoclonal antibodies produced by theseclones (antibodies 36F7C10 (Table 2), 42G5B10 (Table 3), and 40-1C10B9(Table 4)) demonstrated neutralizing activity against human CCL20.

TABLE 2 Amino acid and nucleotide sequences of 36F7C10 DESCRIPTIONSEQUENCE Heavy Chain Signal Sequence (Amino Acid) MRWSCIILFLVATATGVNS(SEQ ID NO: 33) Light Chain Signal Sequence MGVPTQLLLLWLTVVVVRC (AminoAcid) (SEQ ID NO: 34) Heavy Chain Variable Domain Amino AcidQVQLQQPGAELVKPGASVKMSCKASGYT Sequence FTNYWMHWVKQRPGQGLEWIGVIDPSDS (SEQID NO: 39) YTTYNQKFKGKATLTVDTSSSTAYMQLS SLTSEDSAVYYCTRGNYGVDYAMDYWGQGTSVTVSS Light Chain Variable Domain Amino AcidDIQMTQSPASLSASVGETVTITCGASENIY Sequence GALNWYQRKQGKSPQLLIYGATNLADG (SEQID NO: 40) MSSRFSGSGSGRQYSLKISSLHPDDVATY YCQNVLITPYTFGGGTKLEIK HeavyChain Signal Sequence ATGAGATGGAGCTGTATCATCCTCTTCT (Nucleotide)TGGTAGCAACAGCTACAGGTGTCAACT (SEQ ID NO: 45) CC Light Chain SignalSequence ATGGGTGTACCCACTCAGCTCCTGTTGC (Nucleotide)TGTGGCTTACAGTCGTAGTTGTCAGATGT (SEQ ID NO: 46) Heavy Chain VariableDomain Nucleotide CAGGTCCAACTGCAGCAGCCTGGGGCT SequenceGAGCTGGTGAAGCCTGGGGCTTCAGTG (SEQ ID NO: 51) AAGATGTCCTGCAAGGCTTCTGGCTACACCTTCACCAACTACTGGATGCACTGGGT GAAGCAGAGGCCTGGACAAGGCCTTGAGTGGATCGGAGTGATTGATCCTTCTGAT AGTTATACTACCTACAATCAAAAGTTCAAGGGCAAGGCCACATTGACTGTAGACA CATCCTCCAGCACAGCCTACATGCAGCTCAGCAGCCTGACATCTGAGGACTCTGC GGTCTATTACTGTACAAGAGGTAACTACGGAGTAGACTATGCTATGGACTACTGG GGTCAAGGAACCTCAGTCACCGTCTCCT CG Light ChainVariable Domain Nucleotide GACATCCAGATGACTCAGTCTCCAGCTT SequenceCACTGTCTGCATCTGTGGGAGAAACTGT (SEQ ID NO: 52) CACCATCACATGTGGAGCAAGTGAGAATATTTACGGTGCTTTAAATTGGTATCAG CGGAAACAGGGAAAATCTCCTCAGCTCCTGATCTATGGTGCAACCAACTTGGCAG ATGGCATGTCATCGAGGTTCAGTGGCAGTGGATCTGGTAGACAGTATTCTCTCAA GATCAGTAGCCTGCATCCTGACGATGTTGCAACGTATTACTGTCAAAATGTGTTAA TTACTCCGTACACGTTCGGAGGGGGGACCAAGCTGGAAATAAAA

TABLE 3 Amino acid and nucleotide sequences of 42G5B10 DESCRIPTIONSEQUENCE Heavy Chain Signal Sequence (Amino Acid) MRWSCIILFLVATATGVNS(SEQ ID NO: 37) Light Chain Signal Sequence MGVPTQLLLLWLTVVVVRC (AminoAcid) (SEQ ID NO: 38) Heavy Chain Variable Domain Amino AcidQVQLQQPGAELVKPGASVKMSCKASGYT Sequence FTSYWMHWVKQRPGQGLEWIGLIDPSDK (SEQID NO: 43) YTNYNQKFKGKATLTVDTSSSTAYMQLS SLTSEDSAVYYCTRGNYGVDYGMDYWGQGTSVTVSS Light Chain Variable Domain Amino AcidDIQMTQSPASLSASVGETVTITCGASENIY Sequence GALNWYQRKQGKSPQLLIYGATNLADG (SEQID NO: 44) MSSRFSGSGSGRQYSLKISSLHPDDVATY YCQNVLSTPYTFGGGTKLEIK HeavyChain Signal Sequence ATGAGATGGAGCTGTATCATCCTCTTCT (Nucleotide)TGGTAGCAACAGCTACAGGTGTCAACT (SEQ ID NO: 49) CC Light Chain SignalSequence ATGGGTGTACCCACTCAGCTCCTGTTGC (Nucleotide)TGTGGCTTACAGTCGTAGTTGTCAGATGT (SEQ ID NO: 50) Heavy Chain VariableDomain Nucleotide CAGGTCCAACTGCAGCAGCCTGGGGCT SequenceGAGCTGGTGAAGCCTGGGGCTTCAGTG (SEQ ID NO: 55) AAGATGTCCTGCAAGGCTTCTGGCTACACCTTCACCAGCTACTGGATGCACTGGGT GAAGCAGAGGCCTGGACAAGGCCTTGAGTGGATCGGACTGATTGATCCTTCTGAT AAGTATACTAACTACAATCAAAAGTTCAAGGGCAAGGCCACATTGACTGTAGAC ACATCCTCCAGCACAGCCTACATGCAGCTCAGCAGCCTGACATCTGAGGACTCTGC GGTCTATTACTGTACAAGAGGTAACTACGGAGTAGACTATGGTATGGACTACTGG GGTCAAGGAACCTCAGTCACCGTCTCCT CA Light ChainVariable Domain Nucleotide GACATCCAGATGACTCAGTCTCCAGCTT SequenceCACTGTCTGCATCTGTGGGAGAAACTGT (SEQ ID NO: 56) CACCATCACATGTGGAGCAAGTGAGAATATTTACGGTGCTTTAAATTGGTATCAG CGGAAACAGGGAAAATCTCCTCAGCTCCTGATCTATGGTGCAACCAACTTGGCAG ATGGCATGTCATCGAGGTTCAGTGGCAGTGGATCTGGTAGACAGTATTCTCTCAA GATCAGTAGCCTGCATCCTGACGATGTTGCAACGTATTACTGTCAAAATGTGTTAA GTACTCCGTACACGTTCGGAGGGGGGACCAAGCTGGAAATAAAA

TABLE 4 Amino acid and nucleotide sequences of 40-1C10B9 DESCRIPTIONSEQUENCE Heavy Chain Signal Sequence (Amino Acid) MEWSWVFLFLLSVIAGVQS(SEQ ID NO: 35) Light Chain Signal Sequence (Amino Acid)MGVPTQLLLLWLTVVVVRC (SEQ ID NO: 36) Heavy Chain Variable Domain AminoAcid QVQLQQSGAELVRPGASVTLSCKASGYT Sequence FTDYEMHWVKQTPVHGLEWIGAIDPETT(SEQ ID NO: 41) STAYNQKFKGKATLTADKSSSTAYMELR SLTSEDSAVYYCTKCYYGSADYAMDYWGQGTSVTVSS Light Chain Variable Domain Amino AcidDIQMTQSPASLSASVGETVTITCGASENIY Sequence GALNWYQRKQGKSPQLLIYGATNLADG (SEQID NO: 42) MSSRFSGSGSGRQYSLKISSLHPDDVATY YCQNVLSTPWTFGGGTKLEIK HeavyChain Signal Sequence (Nucleotide) ATGGAATGGAGCTGGGTCTTTCTCTTCC (SEQ IDNO: 47) TCCTGTCAGTAATTGCAGGTGTCCAATCC Light Chain Signal Sequence(Nucleotide) ATGGGTGTACCCACTCAGCTCCTGTTGC (SEQ ID NO: 48)TGTGGCTTACAGTCGTAGTTGTCAGATGT Heavy Chain Variable Domain NucleotideCAGGTTCAACTGCAGCAGTCTGGGGCT Sequence GAGCTGGTGAGGCCTGGGGCTTCAGTG (SEQ IDNO: 53) ACGCTGTCCTGCAAGGCTTCGGGCTACA CATTTACTGACTATGAAATGCACTGGGTGAAGCAGACACCTGTGCATGGCCTGGA ATGGATTGGAGCTATTGATCCTGAAACTACTAGTACTGCCTACAATCAGAAGTTCA AGGGCAAGGCCACACTGACTGCAGACAAATCCTCCAGCACAGCCTACATGGAGCT CCGCAGCCTGACATCTGAGGACTCTGCCGTCTATTACTGTACCAAATGTTACTACG GTAGCGCGGACTATGCTATGGACTACTGGGGTCAAGGAACCTCAGTCACCGTCTCC TCA Light Chain Variable Domain NucleotideGACATCCAGATGACTCAGTCTCCAGCTT Sequence CACTGTCTGCATCTGTGGGAGAAACTGT (SEQID NO: 54) CACCATCACATGTGGAGCAAGTGAGAA TATTTACGGTGCTTTAAATTGGTATCAGCGGAAACAGGGAAAATCTCCTCAGCTC CTGATCTATGGTGCAACCAACTTGGCAGATGGCATGTCATCGAGGTTCAGTGGCA GTGGATCTGGTAGACAGTATTCTCTCAAGATCAGTAGCCTGCATCCTGACGATGTT GCAACGTATTACTGTCAAAATGTGTTAAGTACTCCGTGGACGTTCGGTGGAGGCA CCAAGCTGGAAATCAAA

The 36F7C10, 42G5B10, and 40-1C10B9 mouse antibodies were then used toproduce humanized antibody heavy and light chains. The humanizationmethod involved grafting mouse complementarity determining regions(CDRs), as identified by Kabat and/or Chothia definition methodsaccording to Kabat numbering, into the human heavy and light chaingermline sequences representing the best framework matches with theoriginal mouse sequences from which the CDRs were derived (FIGS. 6A-C;bolding denotes residues that differ between the mouse antibody and thehuman germline sequence; underlining and bolding denotes grafted mouseCDRs; italicizing and bolding denotes framework residues substitutedwith corresponding mouse antibody residues; and underlining, italicizingand bolding denotes CDR residues substituted with corresponding humangermline residues). Framework matches were identified using the IgBlastdatabase, according to the methods described in Altschul et al., Nucl.Acids Res. 25:3389-3402 (1997). To derive alternative humanized chainversions, we used two methods: 1) 3D modeling techniques to predictcritical murine residues within the framework interacting with the CDRresidues, and 2) sequence alignments to identify murine residuesimmediately adjacent to the canonical CDR sequences.

The humanized sequences were constructed into expression vectors andtransfected into mammalian cell lines (e.g., HEK293E cells (Invitrogen))for antibody production. Antibodies obtained by this process were thencharacterized in functional and physiochemical assays.

Example 4 In Vitro Chemotaxis Assays Demonstrating NeutralizationActivity of Humanized Anti-Human CCL20 Abs

We identified humanized antibodies that actively neutralize the humanCCL20 ligand by performing in vitro chemotaxis assays usingCCR6-transduced B300.19 cells. Upon determination of IC₅₀, IC₉₀, andIC₉₅ values (FIGS. 7A-C), antibodies demonstrating a reduced value inthe IC₉₅ table were deemed to possess significant neutralizing activity(FIG. 7C). Of the 14 humanized heavy chains and 26 humanized lightchains produced, we tested 41 combinations. Eight of these combinationswere shown to significantly neutralize chemotaxis. These eight humanizedantibodies are listed below:

36LK3/36HKK3

36LK3/42HKK1

36LK3/42HKK2

36LK3/42HKK3

36LK3/36HC2

36LK3/36HC3

36LC3/36HKK3

36LC3/36HC2

The amino acid and nucleotide sequences of humanized heavy chains HC2,HC3, 36HKK3, 42HKK1, 42HKK2, and 42HKK3, and of humanized light chainsLC3 and LK3, are shown in Tables 5-12. The amino acid sequences encodedby the human germline genes utilized by these heavy and light chains areshown in Table 13.

TABLE 5 Amino acid and nucleotide sequences of humanized anti-humanCCL20 antibody heavy chain 36HC2 (“HC2”) SEQUENCE (signal sequenceunderlined, variable domain DESCRIPTION in bold) Heavy Chain Amino AcidSequence MGWSCIILFLVATATGVHS QVQLVQSGA with the signal sequenceEVKKPGASVKVSCKASGYTFTNYWMH (SEQ ID NO: 1) WVRQAPGQGLEWMGVIDPSDSYTTYAQKFQGRVTMTVDTSTSTVYMELSSLRS EDTAVYYCARGNYGVDYAMDYWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTA ALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTY ICNVNHKPSNTKVDKRVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTP EVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQD WLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVK GFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSV MHEALHNHYTQKSLSLSPGK Heavy Chain Amino AcidSequence QVQLVQSGAEVKKPGASVKVSCKASG without the signal sequenceYTFTNYWMHWVRQAPGQGLEWMGVI (SEQ ID NO: 108) DPSDSYTTYAQKFQGRVTMTVDTSTSTVYMELSSLRSEDTAVYYCARGNYGVDY AMDYWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSW NSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKRVE PKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKF NWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALP APIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPE NNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSP GK Heavy Chain Variable Domain Amino AcidQVQLVQSGAEVKKPGASVKVSCKASG Sequence YTFTNYWMHWVRQAPGQGLEWMGVI (SEQ IDNO: 9) DPSDSYTTYAQKFQGRVTMTVDTSTST VYMELSSLRSEDTAVYYCARGNYGVDYAMDYWGQGTLVTVSS Heavy Chain Nucleotide SequenceATGGGCTGGTCCTGCATCATTCTGTTCC with the signal sequenceTGGTGGCCACTGCTACCGGAGTGCACA (SEQ ID NO: 17) GC CAGGTGCAGCTGGTGCAGTCTGGGGCTGAGGTGAAGAAACCCGGTGCAA GTGTGAAGGTGTCATGTAAAGCATCCGGCTATACATTCACTAACTACTGGAT GCATTGGGTGAGGCAGGCTCCAGGACAGGGACTGGAATGGATGGGCGTGA TCGACCCTTCAGATTCCTACACCACATATGCCCAGAAGTTTCAGGGCAGGGT GACCATGACAGTGGACACTAGCACCTCTACAGTGTACATGGAGCTGTCCAGC CTGAGAAGTGAAGATACAGCAGTGTACTATTGCGCCCGCGGCAATTACGGAG TGGACTATGCCATGGATTACTGGGGGCAGGGTACTCTGGTGACCGTGTCTAG TGCTTCTACCAAGGGCCCATCGGTCTTCCCCCTGGCACCCTCCTCCAAGAGCACCT CTGGGGGCACAGCGGCCCTGGGCTGCCTGGTCAAGGACTACTTCCCCGAACCGGT GACGGTGTCGTGGAACTCAGGCGCCCTGACCAGCGGCGTGCACACCTTCCCGGCT GTCCTACAGTCCTCAGGACTCTACTCCCTCAGCAGCGTGGTGACCGTGCCCTCCAG CAGCTTGGGCACCCAGACCTACATCTGCAACGTGAATCACAAGCCCAGCAACACC AAGGTGGACAAGAGAGTTGAGCCCAAATCTTGTGACAAAACTCACACATGCCCAC CGTGCCCAGCACCTGAACTCCTGGGGGGACCGTCAGTCTTCCTCTTCCCCCCAAA ACCCAAGGACACCCTCATGATCTCCCGGACCCCTGAGGTCACATGCGTGGTGGTG GACGTGAGCCACGAAGACCCTGAGGTCAAGTTCAACTGGTACGTGGACGGCGTG GAGGTGCATAATGCCAAGACAAAGCCGCGGGAGGAGCAGTACAACAGCACGTAC CGTGTGGTCAGCGTCCTCACCGTCCTGCACCAGGACTGGCTGAATGGCAAGGAGT ACAAGTGCAAGGTCTCCAACAAAGCCCTCCCAGCCCCCATCGAGAAAACCATCTC CAAAGCCAAAGGGCAGCCCCGAGAACCACAGGTGTACACCCTGCCCCCATCCCGG GAGGAGATGACCAAGAACCAGGTCAGCCTGACCTGCCTGGTCAAAGGCTTCTATC CCAGCGACATCGCCGTGGAGTGGGAGAGCAATGGGCAGCCGGAGAACAACTACA AGACCACGCCTCCCGTGCTGGACTCCGACGGCTCCTTCTTCCTCTATAGCAAGCTC ACCGTGGACAAGAGCAGGTGGCAGCAGGGGAACGTCTTCTCATGCTCCGTGATGC ATGAGGCTCTGCACAACCACTACACGCAGAAGAGCCTCTCCCTGTCTCCCGGGAA ATGA Heavy Chain Nucleotide SequenceCAGGTGCAGCTGGTGCAGTCTGGGG without the signal sequenceCTGAGGTGAAGAAACCCGGTGCAAGT (SEQ ID NO: 109) GTGAAGGTGTCATGTAAAGCATCCGGCTATACATTCACTAACTACTGGATGC ATTGGGTGAGGCAGGCTCCAGGACAGGGACTGGAATGGATGGGCGTGATC GACCCTTCAGATTCCTACACCACATATGCCCAGAAGTTTCAGGGCAGGGTGA CCATGACAGTGGACACTAGCACCTCTACAGTGTACATGGAGCTGTCCAGCCT GAGAAGTGAAGATACAGCAGTGTACTATTGCGCCCGCGGCAATTACGGAGTG GACTATGCCATGGATTACTGGGGGCAGGGTACTCTGGTGACCGTGTCTAGTG CTTCTACCAAGGGCCCATCGGTCTTCCCCCTGGCACCCTCCTCCAAGAGCACCTCT GGGGGCACAGCGGCCCTGGGCTGCCTGGTCAAGGACTACTTCCCCGAACCGGTG ACGGTGTCGTGGAACTCAGGCGCCCTGACCAGCGGCGTGCACACCTTCCCGGCTG TCCTACAGTCCTCAGGACTCTACTCCCTCAGCAGCGTGGTGACCGTGCCCTCCAG CAGCTTGGGCACCCAGACCTACATCTGCAACGTGAATCACAAGCCCAGCAACACC AAGGTGGACAAGAGAGTTGAGCCCAAATCTTGTGACAAAACTCACACATGCCCAC CGTGCCCAGCACCTGAACTCCTGGGGGGACCGTCAGTCTTCCTCTTCCCCCCAAA ACCCAAGGACACCCTCATGATCTCCCGGACCCCTGAGGTCACATGCGTGGTGGTG GACGTGAGCCACGAAGACCCTGAGGTCAAGTTCAACTGGTACGTGGACGGCGTG GAGGTGCATAATGCCAAGACAAAGCCGCGGGAGGAGCAGTACAACAGCACGTAC CGTGTGGTCAGCGTCCTCACCGTCCTGCACCAGGACTGGCTGAATGGCAAGGAGT ACAAGTGCAAGGTCTCCAACAAAGCCCTCCCAGCCCCCATCGAGAAAACCATCTC CAAAGCCAAAGGGCAGCCCCGAGAACCACAGGTGTACACCCTGCCCCCATCCCGG GAGGAGATGACCAAGAACCAGGTCAGCCTGACCTGCCTGGTCAAAGGCTTCTATC CCAGCGACATCGCCGTGGAGTGGGAGAGCAATGGGCAGCCGGAGAACAACTACA AGACCACGCCTCCCGTGCTGGACTCCGACGGCTCCTTCTTCCTCTATAGCAAGCTC ACCGTGGACAAGAGCAGGTGGCAGCAGGGGAACGTCTTCTCATGCTCCGTGATGC ATGAGGCTCTGCACAACCACTACACGCAGAAGAGCCTCTCCCTGTCTCCCGGGAA ATGA Heavy Chain Variable Domain NucleotideCAGGTGCAGCTGGTGCAGTCTGGGG Sequence CTGAGGTGAAGAAACCCGGTGCAAGT (SEQ IDNO: 25) GTGAAGGTGTCATGTAAAGCATCCGG CTATACATTCACTAACTACTGGATGCATTGGGTGAGGCAGGCTCCAGGACA GGGACTGGAATGGATGGGCGTGATCGACCCTTCAGATTCCTACACCACATA TGCCCAGAAGTTTCAGGGCAGGGTGACCATGACAGTGGACACTAGCACCTCT ACAGTGTACATGGAGCTGTCCAGCCTGAGAAGTGAAGATACAGCAGTGTACT ATTGCGCCCGCGGCAATTACGGAGTGGACTATGCCATGGATTACTGGGGGCA GGGTACTCTGGTGACCGTGTCTAGT

TABLE 6 Amino acid and nucleotide sequences of humanized anti-humanCCL20 antibody heavy chain 36HC3 (“HC3”) SEQUENCE (signal sequenceunderlined, variable domain DESCRIPTION in bold) Heavy ChainMGWSCIILFLVATATGVHS QVQLVQSGA Amino Acid SequenceEVKKPGASVKVSCKASGYTFTNYWMH (SEQ ID NO: 2) WVKQAPGQGLEWIGVIDPSDSYTTYNQKFKGKATMTRDTSTSTVYMELSSLRSE DTAVYYCTRGNYGVDYAMDYWGQGTSVTVSSASTKGPSVFPLAPSSKSTSGGTAA LGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYI CNVNHKPSNTKVDKRVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTP EVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQD WLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVK GFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSV MHEALHNHYTQKSLSLSPGK Heavy Chain VariableDomain Amino Acid QVQLVQSGAEVKKPGASVKVSCKASG SequenceYTFTNYWMHWVKQAPGQGLEWIGVID (SEQ ID NO: 10) PSDSYTTYNQKFKGKATMTRDTSTSTVYMELSSLRSEDTAVYYCTRGNYGVDYA MDYWGQGTSVTVSS Heavy ChainATGGGCTGGTCCTGCATCATTCTGTTCC Nucleotide SequenceTGGTGGCAACTGCCACCGGAGTGCACA (SEQ ID NO: 18) GC CAGGTGCAGCTGGTGCAGTCTGGGGCTGAGGTGAAGAAACCCGGTGCAA GTGTGAAAGTGTCATGCAAGGCATCCGGCTATACATTCACTAACTACTGGAT GCATTGGGTGAAGCAGGCACCAGGACAGGGACTGGAATGGATCGGCGTGAT CGACCCTTCAGATTCCTACACCACATATAATCAGAAGTTTAAAGGCAAGGCT ACCATGACAAGGGACACTAGCACCTCTACAGTGTACATGGAGCTGTCCAGCC TGAGGTCCGAAGATACAGCCGTGTACTATTGCACTCGGGGCAACTACGGAGT GGACTATGCTATGGATTACTGGGGGCAGGGTACTAGTGTGACCGTGTCTAGT GCATCTACCAAGGGCCCATCGGTCTTCCCCCTGGCACCCTCCTCCAAGAGCACCTC TGGGGGCACAGCGGCCCTGGGCTGCCTGGTCAAGGACTACTTCCCCGAACCGGT GACGGTGTCGTGGAACTCAGGCGCCCTGACCAGCGGCGTGCACACCTTCCCGGCT GTCCTACAGTCCTCAGGACTCTACTCCCTCAGCAGCGTGGTGACCGTGCCCTCCAG CAGCTTGGGCACCCAGACCTACATCTGCAACGTGAATCACAAGCCCAGCAACACC AAGGTGGACAAGAGAGTTGAGCCCAAATCTTGTGACAAAACTCACACATGCCCAC CGTGCCCAGCACCTGAACTCCTGGGGGGACCGTCAGTCTTCCTCTTCCCCCCAAA ACCCAAGGACACCCTCATGATCTCCCGGACCCCTGAGGTCACATGCGTGGTGGTG GACGTGAGCCACGAAGACCCTGAGGTCAAGTTCAACTGGTACGTGGACGGCGTG GAGGTGCATAATGCCAAGACAAAGCCGCGGGAGGAGCAGTACAACAGCACGTAC CGTGTGGTCAGCGTCCTCACCGTCCTGCACCAGGACTGGCTGAATGGCAAGGAGT ACAAGTGCAAGGTCTCCAACAAAGCCCTCCCAGCCCCCATCGAGAAAACCATCTC CAAAGCCAAAGGGCAGCCCCGAGAACCACAGGTGTACACCCTGCCCCCATCCCGG GAGGAGATGACCAAGAACCAGGTCAGCCTGACCTGCCTGGTCAAAGGCTTCTATC CCAGCGACATCGCCGTGGAGTGGGAGAGCAATGGGCAGCCGGAGAACAACTACA AGACCACGCCTCCCGTGCTGGACTCCGACGGCTCCTTCTTCCTCTATAGCAAGCTC ACCGTGGACAAGAGCAGGTGGCAGCAGGGGAACGTCTTCTCATGCTCCGTGATGC ATGAGGCTCTGCACAACCACTACACGCAGAAGAGCCTCTCCCTGTCTCCCGGGAA ATGA Heavy Chain Variable Domain NucleotideCAGGTGCAGCTGGTGCAGTCTGGGG Sequence CTGAGGTGAAGAAACCCGGTGCAAGT (SEQ IDNO: 26) GTGAAAGTGTCATGCAAGGCATCCGG CTATACATTCACTAACTACTGGATGCATTGGGTGAAGCAGGCACCAGGACA GGGACTGGAATGGATCGGCGTGATCGACCCTTCAGATTCCTACACCACATA TAATCAGAAGTTTAAAGGCAAGGCTACCATGACAAGGGACACTAGCACCTCT ACAGTGTACATGGAGCTGTCCAGCCTGAGGTCCGAAGATACAGCCGTGTACT ATTGCACTCGGGGCAACTACGGAGTGGACTATGCTATGGATTACTGGGGGCA GGGTACTAGTGTGACCGTGTCTAGT

TABLE 7 Amino acid and nucleotide sequences of humanized anti-humanCCL20 antibody heavy chain 36HKK3 SEQUENCE (signal sequence underlined,variable domain DESCRIPTION in bold) Heavy Chain MDWTWRILFLVAAATGAHSQVQLVQSG Amino Acid Sequence AEVKKPGASVKVSCKASGYTFTNYWM (SEQ ID NO: 3)HWVRQAPGQGLEWMGVIDPSDSYTTY NQKFKGKATLTVDTSTSTAYMELSSLRSEDTAVYYCTRGNYGVDYAMDYWGQ GTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGV HTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKRVEPKSCDKTHT CPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVE VHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAK GQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVL DSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK Heavy Chain Variable Domain Amino AcidQVQLVQSGAEVKKPGASVKVSCKASG Sequence YTFTNYWMHWVRQAPGQGLEWMGVI (SEQ IDNO: 11) DPSDSYTTYNQKFKGKATLTVDTSTST AYMELSSLRSEDTAVYYCTRGNYGVDYAMDYWGQGTLVTVSS Heavy Chain ATGGACTGGACATGGAGAATCCTGTTCC NucleotideSequence TGGTGGCCGCTGCAACCGGAGCACACA (SEQ ID NO: 19) GCCAGGTGCAGCTGGTGCAGTCTGGA GCAGAGGTGAAGAAACCCGGTGCTAGTGTGAAAGTGTCATGCAAGGCCTCC GGGTATACTTTCACCAACTACTGGATGCATTGGGTGAGGCAGGCTCCAGGA CAGGGACTGGAATGGATGGGCGTGATTGACCCTTCAGATTCCTACACCACA TATAATCAGAAGTTTAAAGGAAAGGCAACACTGACTGTGGACACCAGCACAT CTACTGCCTACATGGAGCTGTCCAGCCTGAGGTCCGAAGATACTGCCGTGTA CTATTGTACCCGGGGCAACTACGGAGTGGACTATGCAATGGATTACTGGGGG CAGGGTACCCTGGTGACAGTGTCTAGTGCTAGCACCAAGGGCCCATCGGTCTTC CCCCTGGCACCCTCCTCCAAGAGCACCTCTGGGGGCACAGCGGCCCTGGGCTGCC TGGTCAAGGACTACTTCCCCGAACCGGTGACGGTGTCGTGGAACTCAGGCGCCCT GACCAGCGGCGTGCACACCTTCCCGGCTGTCCTACAGTCCTCAGGACTCTACTCCC TCAGCAGCGTGGTGACCGTGCCCTCCAGCAGCTTGGGCACCCAGACCTACATCTGC AACGTGAATCACAAGCCCAGCAACACCAAGGTGGACAAGAGAGTTGAGCCCAAA TCTTGTGACAAAACTCACACATGCCCACCGTGCCCAGCACCTGAACTCCTGGGGG GACCGTCAGTCTTCCTCTTCCCCCCAAAACCCAAGGACACCCTCATGATCTCCCGG ACCCCTGAGGTCACATGCGTGGTGGTGGACGTGAGCCACGAAGACCCTGAGGTC AAGTTCAACTGGTACGTGGACGGCGTGGAGGTGCATAATGCCAAGACAAAGCCG CGGGAGGAGCAGTACAACAGCACGTACCGTGTGGTCAGCGTCCTCACCGTCCTGC ACCAGGACTGGCTGAATGGCAAGGAGTACAAGTGCAAGGTCTCCAACAAAGCCC TCCCAGCCCCCATCGAGAAAACCATCTCCAAAGCCAAAGGGCAGCCCCGAGAACC ACAGGTGTACACCCTGCCCCCATCCCGGGAGGAGATGACCAAGAACCAGGTCAGC CTGACCTGCCTGGTCAAAGGCTTCTATCCCAGCGACATCGCCGTGGAGTGGGAGA GCAATGGGCAGCCGGAGAACAACTACAAGACCACGCCTCCCGTGCTGGACTCCGA CGGCTCCTTCTTCCTCTATAGCAAGCTCACCGTGGACAAGAGCAGGTGGCAGCAG GGGAACGTCTTCTCATGCTCCGTGATGCATGAGGCTCTGCACAACCACTACACGC AGAAGAGCCTCTCCCTGTCTCCGGGTAA ATGA HeavyChain Variable Domain Nucleotide CAGGTGCAGCTGGTGCAGTCTGGAGC SequenceAGAGGTGAAGAAACCCGGTGCTAGTG (SEQ ID NO: 27) TGAAAGTGTCATGCAAGGCCTCCGGGTATACTTTCACCAACTACTGGATGCA TTGGGTGAGGCAGGCTCCAGGACAGGGACTGGAATGGATGGGCGTGATTGA CCCTTCAGATTCCTACACCACATATAATCAGAAGTTTAAAGGAAAGGCAACA CTGACTGTGGACACCAGCACATCTACTGCCTACATGGAGCTGTCCAGCCTGA GGTCCGAAGATACTGCCGTGTACTATTGTACCCGGGGCAACTACGGAGTGGA CTATGCAATGGATTACTGGGGGCAGGGTACCCTGGTGACAGTGTCTAGT

TABLE 8 Amino acid and nucleotide sequences of humanized anti-humanCCL20 antibody heavy chain 42HKK1 SEQUENCE (signal sequence underlined,variable domain DESCRIPTION in bold) Heavy Chain MDWTWRILFLVAAATGAHSQVQLVQSG Amino Acid Sequence AEVKKPGASVKVSCKASGYTFTSYWM (SEQ ID NO: 4)HWVRQAPGQGLEWMGLIDPSDKYTNY NQKFKGRVTMTRDTSTSTVYMELSSLRSEDTAVYYCARGNYGVDYGMDYWGQ GTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGV HTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKRVEPKSCDKTHT CPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVE VHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAK GQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVL DSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK Heavy Chain Variable Domain Amino AcidQVQLVQSGAEVKKPGASVKVSCKASG Sequence YTFTSYWMHWVRQAPGQGLEWMGLI (SEQ IDNO: 12) DPSDKYTNYNQKFKGRVTMTRDTSTST VYMELSSLRSEDTAVYYCARGNYGVDYGMDYWGQGTLVTVSS Heavy Chain ATGGACTGGACCTGGCGAATCCTGTTCC NucleotideSequence TGGTGGCCGCTGCAACAGGAGCACACT (SEQ ID NO: 20) CACAGGTGCAGCTGGTGCAGTCCGGG GCAGAGGTGAAGAAACCCGGTGCCAGCGTGAAGGTGTCTTGCAAAGCTAGT GGCTATACCTTCACAAGCTACTGGATGCATTGGGTGCGGCAGGCACCAGGA CAGGGACTGGAATGGATGGGCCTGATTGACCCTTCTGATAAGTACACTAACT ACAACCAGAAGTTTAAAGGAAGGGTGACTATGACCCGGGACACATCAACTTC CACCGTGTACATGGAGCTGTCCAGCCTGAGATCCGAAGATACCGCCGTGTAC TATTGTGCTCGCGGCAACTACGGAGTGGACTATGGCATGGATTACTGGGGGC AGGGTACACTGGTGACCGTGTCCAGTGCTAGCACCAAGGGCCCATCGGTCTTCC CCCTGGCACCCTCCTCCAAGAGCACCTCTGGGGGCACAGCGGCCCTGGGCTGCCT GGTCAAGGACTACTTCCCCGAACCGGTGACGGTGTCGTGGAACTCAGGCGCCCT GACCAGCGGCGTGCACACCTTCCCGGCTGTCCTACAGTCCTCAGGACTCTACTCCC TCAGCAGCGTGGTGACCGTGCCCTCCAGCAGCTTGGGCACCCAGACCTACATCTGC AACGTGAATCACAAGCCCAGCAACACCAAGGTGGACAAGAGAGTTGAGCCCAAA TCTTGTGACAAAACTCACACATGCCCACCGTGCCCAGCACCTGAACTCCTGGGGG GACCGTCAGTCTTCCTCTTCCCCCCAAAACCCAAGGACACCCTCATGATCTCCCGG ACCCCTGAGGTCACATGCGTGGTGGTGGACGTGAGCCACGAAGACCCTGAGGTC AAGTTCAACTGGTACGTGGACGGCGTGGAGGTGCATAATGCCAAGACAAAGCCG CGGGAGGAGCAGTACAACAGCACGTACCGTGTGGTCAGCGTCCTCACCGTCCTGC ACCAGGACTGGCTGAATGGCAAGGAGTACAAGTGCAAGGTCTCCAACAAAGCCC TCCCAGCCCCCATCGAGAAAACCATCTCCAAAGCCAAAGGGCAGCCCCGAGAACC ACAGGTGTACACCCTGCCCCCATCCCGGGAGGAGATGACCAAGAACCAGGTCAGC CTGACCTGCCTGGTCAAAGGCTTCTATCCCAGCGACATCGCCGTGGAGTGGGAGA GCAATGGGCAGCCGGAGAACAACTACAAGACCACGCCTCCCGTGCTGGACTCCGA CGGCTCCTTCTTCCTCTATAGCAAGCTCACCGTGGACAAGAGCAGGTGGCAGCAG GGGAACGTCTTCTCATGCTCCGTGATGCATGAGGCTCTGCACAACCACTACACGC AGAAGAGCCTCTCCCTGTCTCCGGGTAA ATGA HeavyChain Variable Domain Nucleotide CAGGTGCAGCTGGTGCAGTCCGGGG SequenceCAGAGGTGAAGAAACCCGGTGCCAG (SEQ ID NO: 28) CGTGAAGGTGTCTTGCAAAGCTAGTGGCTATACCTTCACAAGCTACTGGATG CATTGGGTGCGGCAGGCACCAGGACAGGGACTGGAATGGATGGGCCTGATT GACCCTTCTGATAAGTACACTAACTACAACCAGAAGTTTAAAGGAAGGGTGA CTATGACCCGGGACACATCAACTTCCACCGTGTACATGGAGCTGTCCAGCCT GAGATCCGAAGATACCGCCGTGTACTATTGTGCTCGCGGCAACTACGGAGTG GACTATGGCATGGATTACTGGGGGCAGGGTACACTGGTGACCGTGTCCAGT

TABLE 9 Amino acid and nucleotide sequences of humanized anti-humanCCL20 antibody heavy chain 42HKK2 SEQUENCE (signal sequence underlined,variable domain DESCRIPTION in bold) Heavy Chain MDWTWRILFLVAAATGAHSQVQLVQSG Amino Acid Sequence AEVKKPGASVKVSCKASGYTFTSYWM (SEQ ID NO: 5)HWVRQAPGQGLEWMGLIDPSDKYTNY NQKFKGRVTLTVDTSTSTVYMELSSLRSEDTAVYYCTRGNYGVDYGMDYWGQ GTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGV HTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKRVEPKSCDKTHT CPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVE VHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAK GQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVL DSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK Heavy Chain Variable Domain Amino AcidQVQLVQSGAEVKKPGASVKVSCKASG Sequence YTFTSYWMHWVRQAPGQGLEWMGLI (SEQ IDNO: 13) DPSDKYTNYNQKFKGRVTLTVDTSTST VYMELSSLRSEDTAVYYCTRGNYGVDYGMDYWGQGTLVTVSS Heavy Chain ATGGACTGGACTTGGAGGATCCTGTTCC NucleotideSequence TGGTGGCCGCTGCAACCGGAGCTCACTC (SEQ ID NO: 21) ACAGGTGCAGCTGGTGCAGTCCGGA GCAGAGGTGAAGAAACCCGGTGCCTCCGTGAAGGTGTCTTGCAAAGCAAGTG GCTATACCTTCACAAGCTACTGGATGCATTGGGTGAGACAGGCACCAGGACA GGGACTGGAATGGATGGGCCTGATTGACCCTTCTGATAAGTACACCAACTAC AACCAGAAGTTTAAAGGACGCGTGACTCTGACCGTGGACACATCAACTTCCA CCGTGTACATGGAGCTGTCCAGCCTGAGGTCCGAAGATACCGCAGTGTACTA TTGTACACGGGGCAACTACGGAGTGGACTATGGCATGGATTACTGGGGGCAG GGTACACTGGTGACCGTGTCCAGTGCTAGCACCAAGGGCCCATCGGTCTTCCCC CTGGCACCCTCCTCCAAGAGCACCTCTGGGGGCACAGCGGCCCTGGGCTGCCTGG TCAAGGACTACTTCCCCGAACCGGTGACGGTGTCGTGGAACTCAGGCGCCCTGAC CAGCGGCGTGCACACCTTCCCGGCTGTCCTACAGTCCTCAGGACTCTACTCCCTCA GCAGCGTGGTGACCGTGCCCTCCAGCAGCTTGGGCACCCAGACCTACATCTGCAA CGTGAATCACAAGCCCAGCAACACCAAGGTGGACAAGAGAGTTGAGCCCAAATC TTGTGACAAAACTCACACATGCCCACCGTGCCCAGCACCTGAACTCCTGGGGGGA CCGTCAGTCTTCCTCTTCCCCCCAAAACCCAAGGACACCCTCATGATCTCCCGGAC CCCTGAGGTCACATGCGTGGTGGTGGACGTGAGCCACGAAGACCCTGAGGTCAA GTTCAACTGGTACGTGGACGGCGTGGAGGTGCATAATGCCAAGACAAAGCCGCG GGAGGAGCAGTACAACAGCACGTACCGTGTGGTCAGCGTCCTCACCGTCCTGCAC CAGGACTGGCTGAATGGCAAGGAGTACAAGTGCAAGGTCTCCAACAAAGCCCTC CCAGCCCCCATCGAGAAAACCATCTCCAAAGCCAAAGGGCAGCCCCGAGAACCA CAGGTGTACACCCTGCCCCCATCCCGGGAGGAGATGACCAAGAACCAGGTCAGCC TGACCTGCCTGGTCAAAGGCTTCTATCCCAGCGACATCGCCGTGGAGTGGGAGAG CAATGGGCAGCCGGAGAACAACTACAAGACCACGCCTCCCGTGCTGGACTCCGAC GGCTCCTTCTTCCTCTATAGCAAGCTCACCGTGGACAAGAGCAGGTGGCAGCAGG GGAACGTCTTCTCATGCTCCGTGATGCATGAGGCTCTGCACAACCACTACACGCA GAAGAGCCTCTCCCTGTCTCCGGGTAAA TGA Heavy ChainVariable Domain Nucleotide CAGGTGCAGCTGGTGCAGTCCGGAG SequenceCAGAGGTGAAGAAACCCGGTGCCTCC (SEQ ID NO: 29) GTGAAGGTGTCTTGCAAAGCAAGTGGCTATACCTTCACAAGCTACTGGATGC ATTGGGTGAGACAGGCACCAGGACAGGGACTGGAATGGATGGGCCTGATTG ACCCTTCTGATAAGTACACCAACTACAACCAGAAGTTTAAAGGACGCGTGAC TCTGACCGTGGACACATCAACTTCCACCGTGTACATGGAGCTGTCCAGCCTG AGGTCCGAAGATACCGCAGTGTACTATTGTACACGGGGCAACTACGGAGTGG ACTATGGCATGGATTACTGGGGGCAGGGTACACTGGTGACCGTGTCCAGT

TABLE 10 Amino acid and nucleotide sequences of humanized anti-humanCCL20 antibody heavy chain 42HKK3 SEQUENCE (signal sequence underlined,variable domain DESCRIPTION in bold) Heavy Chain MDWTWRILFLVAAATGAHSQVQLVQSG Amino Acid Sequence AEVKKPGASVKVSCKASGYTFTSYWM (SEQ ID NO: 6)HWVRQAPGQGLEWMGLIDPSDKYTNY NQKFKGKATLTVDTSTSTAYMELSSLRSEDTAVYYCTRGNYGVDYGMDYWGQ GTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGV HTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKRVEPKSCDKTHT CPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVE VHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAK GQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVL DSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK Heavy Chain Variable Domain Amino AcidQVQLVQSGAEVKKPGASVKVSCKASG Sequence YTFTSYWMHWVRQAPGQGLEWMGLI (SEQ IDNO: 14) DPSDKYTNYNQKFKGKATLTVDTSTST AYMELSSLRSEDTAVYYCTRGNYGVDYGMDYWGQGTLVTVSS Heavy Chain ATGGACTGGACTTGGAGAATCCTGTTCC NucleotideSequence TGGTGGCCGCTGCAACCGGAGCTCACTC (SEQ ID NO: 22) ACAGGTGCAGCTGGTGCAGTCCGGA GCAGAGGTGAAGAAACCCGGTGCCTCCGTGAAAGTGTCTTGCAAGGCTAGTG GCTATACCTTCACAAGCTACTGGATGCATTGGGTGAGGCAGGCACCAGGAC AGGGACTGGAATGGATGGGCCTGATTGACCCTTCTGATAAGTACACCAACTA CAACCAGAAGTTTAAAGGAAAGGCAACTCTGACCGTGGACACATCAACTTCC ACCGCCTACATGGAGCTGTCCAGCCTGAGGTCCGAAGATACCGCCGTGTACT ATTGTACACGGGGCAACTACGGAGTGGACTATGGCATGGATTACTGGGGGCA GGGTACACTGGTGACCGTGTCCAGTGCTAGCACCAAGGGCCCATCGGTCTTCCC CCTGGCACCCTCCTCCAAGAGCACCTCTGGGGGCACAGCGGCCCTGGGCTGCCTG GTCAAGGACTACTTCCCCGAACCGGTGACGGTGTCGTGGAACTCAGGCGCCCTG ACCAGCGGCGTGCACACCTTCCCGGCTGTCCTACAGTCCTCAGGACTCTACTCCCT CAGCAGCGTGGTGACCGTGCCCTCCAGCAGCTTGGGCACCCAGACCTACATCTGC AACGTGAATCACAAGCCCAGCAACACCAAGGTGGACAAGAGAGTTGAGCCCAAA TCTTGTGACAAAACTCACACATGCCCACCGTGCCCAGCACCTGAACTCCTGGGGG GACCGTCAGTCTTCCTCTTCCCCCCAAAACCCAAGGACACCCTCATGATCTCCCGG ACCCCTGAGGTCACATGCGTGGTGGTGGACGTGAGCCACGAAGACCCTGAGGTC AAGTTCAACTGGTACGTGGACGGCGTGGAGGTGCATAATGCCAAGACAAAGCCG CGGGAGGAGCAGTACAACAGCACGTACCGTGTGGTCAGCGTCCTCACCGTCCTGC ACCAGGACTGGCTGAATGGCAAGGAGTACAAGTGCAAGGTCTCCAACAAAGCCC TCCCAGCCCCCATCGAGAAAACCATCTCCAAAGCCAAAGGGCAGCCCCGAGAACC ACAGGTGTACACCCTGCCCCCATCCCGGGAGGAGATGACCAAGAACCAGGTCAGC CTGACCTGCCTGGTCAAAGGCTTCTATCCCAGCGACATCGCCGTGGAGTGGGAGA GCAATGGGCAGCCGGAGAACAACTACAAGACCACGCCTCCCGTGCTGGACTCCGA CGGCTCCTTCTTCCTCTATAGCAAGCTCACCGTGGACAAGAGCAGGTGGCAGCAG GGGAACGTCTTCTCATGCTCCGTGATGCATGAGGCTCTGCACAACCACTACACGC AGAAGAGCCTCTCCCTGTCTCCGGGTAA ATGA HeavyChain Variable Domain Nucleotide CAGGTGCAGCTGGTGCAGTCCGGAG SequenceCAGAGGTGAAGAAACCCGGTGCCTCC (SEQ ID NO: 30) GTGAAAGTGTCTTGCAAGGCTAGTGGCTATACCTTCACAAGCTACTGGATGC ATTGGGTGAGGCAGGCACCAGGACAGGGACTGGAATGGATGGGCCTGATTG ACCCTTCTGATAAGTACACCAACTACAACCAGAAGTTTAAAGGAAAGGCAAC TCTGACCGTGGACACATCAACTTCCACCGCCTACATGGAGCTGTCCAGCCTG AGGTCCGAAGATACCGCCGTGTACTATTGTACACGGGGCAACTACGGAGTGG ACTATGGCATGGATTACTGGGGGCAGGGTACACTGGTGACCGTGTCCAGT

TABLE 11 Amino acid and nucleotide sequences of humanized anti-humanCCL20 antibody light chain LC3 SEQUENCE (signal sequence underlined,variable domain DESCRIPTION in bold) Light Chain Amino Acid SequenceMGWSCIILFLVATATGVHS DIQMTQSPSS with the signal sequenceLSASVGDRVTITCRASENIYGALNWYQ (SEQ ID NO: 7) QKPGKAPKLLIYGATNLADGVPSRFSGSGSGRQYSLTISSLQPEDFATYYCQNVL ITPYTFGGGTKLEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKV DNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTK SFNRGEC Light Chain Amino Acid SequenceDIQMTQSPSSLSASVGDRVTITCRASENI without the signal sequenceYGALNWYQQKPGKAPKLLIYGATNLA (SEQ ID NO: 110)DGVPSRFSGSGSGRQYSLTISSLQPEDFA TYYCQNVLITPYTFGGGTKLEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPR EAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTH QGLSSPVTKSFNRGEC Light Chain VariableDomain Amino Acid DIQMTQSPSSLSASVGDRVTITCRASENI SequenceYGALNWYQQKPGKAPKLLIYGATNLA (SEQ ID NO: 15) DGVPSRFSGSGSGRQYSLTISSLQPEDFATYYCQNVLITPYTFGGGTKLEIK Light Chain Nucleotide SequenceATGGGCTGGTCCTGCATCATTCTGTTCC with the signal sequenceTGGTGGCAACCGCCACAGGAGTGCACA (SEQ ID NO: 23) GC GACATCCAGATGACCCAGTCTCCATCCAGCCTGAGTGCCTCAGTGGGCGA TAGGGTGACTATCACCTGTCGGGCCAGCGAGAACATCTACGGCGCTCTGAAT TGGTATCAGCAGAAGCCAGGAAAAGCTCCCAAGCTGCTGATCTACGGGGCTA CAAACCTGGCAGACGGTGTGCCCAGTCGATTCTCCGGTAGCGGCTCTGGACG ACAGTATTCACTGACTATCTCTAGTCTGCAGCCTGAAGATTTCGCCACTTACT ATTGCCAGAATGTGCTGATTACTCCATATACCTTTGGCGGAGGGACAAAACT GGAGATCAAGAGAACTGTGGCCGCTCCCAGTGTGTTCATTTTTCCCCCTTCAGAC GAACAGCTGAAATCAGGGACCGCTTCCGTGGTGTGTCTGCTGAACAATTTCTACC CTCGCGAGGCAAAAGTGCAGTGGAAGGTGGATAACGCCCTGCAGAGTGGCAATT CACAGGAGTCCGTGACCGAACAGGACAGCAAAGATTCTACATATAGTCTGTCATC CACCCTGACACTGAGCAAGGCTGATTACGAGAAGCACAAAGTGTATGCATGCGA AGTGACTCATCAGGGGCTGAGCTCTCCCGTGACCAAGTCTTTTAACCGGGGTGAAT GTTGA Light Chain Nucleotide SequenceGACATCCAGATGACCCAGTCTCCATC without the signal sequenceCAGCCTGAGTGCCTCAGTGGGCGATA (SEQ ID NO: 111) GGGTGACTATCACCTGTCGGGCCAGCGAGAACATCTACGGCGCTCTGAATTG GTATCAGCAGAAGCCAGGAAAAGCTCCCAAGCTGCTGATCTACGGGGCTACA AACCTGGCAGACGGTGTGCCCAGTCGATTCTCCGGTAGCGGCTCTGGACGAC AGTATTCACTGACTATCTCTAGTCTGCAGCCTGAAGATTTCGCCACTTACTA TTGCCAGAATGTGCTGATTACTCCATATACCTTTGGCGGAGGGACAAAACTG GAGATCAAGAGAACTGTGGCCGCTCCCAGTGTGTTCATTTTTCCCCCTTCAGACG AACAGCTGAAATCAGGGACCGCTTCCGTGGTGTGTCTGCTGAACAATTTCTACCC TCGCGAGGCAAAAGTGCAGTGGAAGGTGGATAACGCCCTGCAGAGTGGCAATTC ACAGGAGTCCGTGACCGAACAGGACAGCAAAGATTCTACATATAGTCTGTCATCC ACCCTGACACTGAGCAAGGCTGATTACGAGAAGCACAAAGTGTATGCATGCGAA GTGACTCATCAGGGGCTGAGCTCTCCCGTGACCAAGTCTTTTAACCGGGGTGAATG TTGA Light Chain Variable Domain NucleotideGACATCCAGATGACCCAGTCTCCATC Sequence CAGCCTGAGTGCCTCAGTGGGCGATA (SEQ IDNO: 31) GGGTGACTATCACCTGTCGGGCCAGC GAGAACATCTACGGCGCTCTGAATTGGTATCAGCAGAAGCCAGGAAAAGCTC CCAAGCTGCTGATCTACGGGGCTACAAACCTGGCAGACGGTGTGCCCAGTCG ATTCTCCGGTAGCGGCTCTGGACGACAGTATTCACTGACTATCTCTAGTCTG CAGCCTGAAGATTTCGCCACTTACTATTGCCAGAATGTGCTGATTACTCCAT ATACCTTTGGCGGAGGGACAAAACTG GAGATCAAG

TABLE 12 Amino acid and nucleotide sequences of humanized anti-humanCCL20 antibody light chain LK3 SEQUENCE (signal sequence underlined,variable domain DESCRIPTION in bold) Light Chain Amino Acid SequenceMDMRVPAQLLGLLLLWLRGARC DIQMT with the signal sequenceQSPSSLSASVGDRVTITCGASENIYGALN (SEQ ID NO: 8) WYQRKPGKAPKLLIYGATNLADGVPSRFSGSGSGRDYTLTISSLQPEDFATYYCQ NVLITPYTFGQGTKLEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQ WKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSP VTKSFNRGEC Light Chain Amino Acid SequenceDIQMTQSPSSLSASVGDRVTITCGASENI without the signal sequenceYGALNWYQRKPGKAPKLLIYGATNLA (SEQ ID NO: 112) DGVPSRFSGSGSGRDYTLTISSLQPEDFATYYCQNVLITPYTFGQGTKLEIKRTVA APSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDS KDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC Light Chain Variable Domain Amino AcidDIQMTQSPSSLSASVGDRVTITCGASENI Sequence YGALNWYQRKPGKAPKLLIYGATNLA (SEQID NO: 16) DGVPSRFSGSGSGRDYTLTISSLQPEDF ATYYCQNVLITPYTFGQGTKLEIK LightChain Nucleotide Sequence ATGGACATGAGGGTGCCTGCTCAGCTG with the signalsequence CTGGGACTGCTGCTGCTGTGGCTGAGGG (SEQ ID NO: 24) GAGCACGATGCGACATCCAGATGACT CAGAGCCCATCCAGCCTGTCAGCCTC CGTGGGCGACAGGGTGACCATCACATGTGGAGCATCCGAGAACATCTACGGG GCCCTGAATTGGTATCAGAGGAAGCCCGGCAAAGCTCCTAAGCTGCTGATCT ACGGTGCCACAAACCTGGCTGATGGCGTGCCCTCCAGATTCAGCGGCTCTGG AAGTGGGCGCGACTATACTCTGACCATTTCTAGTCTGCAGCCAGAGGATTTC GCCACCTACTATTGCCAGAATGTGCTGATCACACCCTACACTTTTGGTCAGG GCACAAAACTGGAAATTAAGCGTACGGTGGCTGCACCATCTGTCTTCATCTTCC CGCCATCTGATGAGCAGTTGAAATCTGGAACTGCCTCTGTTGTGTGCCTGCTGAAT AACTTCTATCCCAGAGAGGCCAAAGTACAGTGGAAGGTGGATAACGCCCTCCAA TCGGGTAACTCCCAGGAGAGTGTCACAGAGCAGGACAGCAAGGACAGCACCTAC AGCCTCAGCAGCACCCTGACGCTGAGCAAAGCAGACTACGAGAAACACAAAGTC TACGCCTGCGAAGTCACCCATCAGGGCCTGAGCTCGCCCGTCACAAAGAGCTTCA ACAGGGGAGAGTGTTGA Light Chain NucleotideSequence GACATCCAGATGACTCAGAGCCCATC without the signal sequenceCAGCCTGTCAGCCTCCGTGGGCGACA (SEQ ID NO: 113) GGGTGACCATCACATGTGGAGCATCCGAGAACATCTACGGGGCCCTGAATTG GTATCAGAGGAAGCCCGGCAAAGCTCCTAAGCTGCTGATCTACGGTGCCACA AACCTGGCTGATGGCGTGCCCTCCAGATTCAGCGGCTCTGGAAGTGGGCGCG ACTATACTCTGACCATTTCTAGTCTGCAGCCAGAGGATTTCGCCACCTACTAT TGCCAGAATGTGCTGATCACACCCTACACTTTTGGTCAGGGCACAAAACTGG AAATTAAGCGTACGGTGGCTGCACCATCTGTCTTCATCTTCCCGCCATCTGATGA GCAGTTGAAATCTGGAACTGCCTCTGTTGTGTGCCTGCTGAATAACTTCTATCCCA GAGAGGCCAAAGTACAGTGGAAGGTGGATAACGCCCTCCAATCGGGTAACTCCCA GGAGAGTGTCACAGAGCAGGACAGCAAGGACAGCACCTACAGCCTCAGCAGCAC CCTGACGCTGAGCAAAGCAGACTACGAGAAACACAAAGTCTACGCCTGCGAAGT CACCCATCAGGGCCTGAGCTCGCCCGTCACAAAGAGCTTCAACAGGGGAGAGTGT TGA Light Chain Variable Domain NucleotideGACATCCAGATGACTCAGAGCCCATC Sequence CAGCCTGTCAGCCTCCGTGGGCGACA (SEQ IDNO: 32) GGGTGACCATCACATGTGGAGCATCC GAGAACATCTACGGGGCCCTGAATTGGTATCAGAGGAAGCCCGGCAAAGCTC CTAAGCTGCTGATCTACGGTGCCACAAACCTGGCTGATGGCGTGCCCTCCAG ATTCAGCGGCTCTGGAAGTGGGCGCGACTATACTCTGACCATTTCTAGTCTGC AGCCAGAGGATTTCGCCACCTACTATTGCCAGAATGTGCTGATCACACCCTA CACTTTTGGTCAGGGCACAAAACTGG AAATTAAG

TABLE 13 Amino acid sequences encoded by human germline genesDESCRIPTION SEQUENCE IGHV1-46*03 QVQLVQSGAEVKKPGASVKVSCKASGYT (SEQ IDNO: 57) FTSYYMHWVRQAPGQGLEWMGIINPSGG STSYAQKFQGRVTMTRDTSTSTVYMELSSLRSEDTAVYYCAR JH4 WGQGTLVTVSS (SEQ ID NO: 117) IGKV1D-39*01DIQMTQSPSSLSASVGDRVTITCRASQSISS (SEQ ID NO: 59)YLNWYQQKPGKAPKLLIYAASSLQSGVP SRFSGSGSGTDFTLTISSLQPEDFATYYCQ QSYSTPP

Based on the evaluation results, we selected 36LK3/36HC2 (“HC2/LK3”) and36LC3/36HC2 (“HC2/LC3”) for further studies. For these two antibodies,in vitro chemotaxis assays were performed in parallel with parentalmouse clone 36F7C10 and its chimeric form (comprising a human Fcportion). In this assay, we employed transwell culture plates withB300.19 CCR6+ cells seeded in the upper layer and recombinant humanCCL20 ligand in the lower layer. Recombinant human CCL20 (10 nM final,R&D Systems) was pre-incubated with the humanized anti-CCL20 antibodiesat room temperature. After 30 min, human CCR6-transduced murine pre-Bcells (B300.19, provided by Dr. H. Kawasaki at Tokyo University) wereapplied to the upper layer and chemotaxis was developed at 37° C. for 4hrs. At the end of incubation, FACS was used to measure migrated cells.The 50%, 90%, and 95% inhibitory concentrations (IC₅₀, IC₉₀, and IC₉₅,respectively) for HC2/LK3 and HC2/LC3 were then calculated (Table 14).Neither of the humanized antibodies lost neutralization activity incomparison with the parental mouse MAb, and IC₅₀ values were calculatedat around 1 nM.

TABLE 14 Neutralizing activity of HC2/LK3 and HC2/LC3 against humanCCL20 in a chemotaxis assay (in nM) Mouse MAb Chimeric MAb IC₅₀ 1.697 ±0.464 1.438 ± 0.216 IC₉₀ 3.944 ± 0.593 3.510 ± 0.601 IC₉₅ 7.680 ± 2.9665.480 ± 1.179 Assay-1 -2 -3 Average HuMAb HC2/LK3 IC₅₀ 0.65 1.09 1.301.014 ± 0.330 IC₉₀ 3.55 3.35 6.79 4.564 ± 1.928 IC₉₅ 7.59 5.41 13.918.972 ± 4.417 HuMAb HC2/LC3 IC₅₀ 0.90 1.26 1.20 1.117 ± 0.193 IC₉₀ 4.645.82 5.30 5.251 ± 0.591 IC₉₅ 11.77 >66.77 10.33 —The dose responses for a representative trial with the HC2/LK3 antibodyare shown in FIGS. 8A-C. Three independent experiments are depicted.

We further confirmed the neutralizing activity of HC2/LK3 and HC2/LC3using a transendothelial migration (TEM) assay in which freshly isolatedhuman peripheral blood mononuclear cells were used instead ofCCR6-transduced artificial cells. Because CD3+CD4+CD45RO+ memory T cellsand CD19+ B cells are well known to be enriched with CCR6-positivecells, we measured the migrated cell numbers of these populations in thepresence or absence of the HC2/LK3 and HC2/LC3 antibodies, as well asthe parental mouse antibody 36F7C10. Both humanized antibodies (FIGS. 9Band C) demonstrated dose dependent inhibition of cell migrationcomparable to that of 36F7C10 (FIG. 9A), providing further evidence oftheir neutralizing activity.

Example 5 Binding of Humanized Anti-Human CCL20 MAbs to Human CCL20

To measure the binding affinity of humanized antibodies HC2/LC3 andHC2/LK3 by surface plasmon resonance (Biacore™), we expressed andpurified the antibodies from conditioned media oftransiently-transfected HEK293F cells, and captured the antibodies on aCM5 chip coated with anti-human Fc monoclonal antibodies using HBS-EP asthe running buffer. We then injected several dilutions (100, 20, 4, 0.8,0.16, and 0 nM) of human CCL20 protein (R&D Systems) over the coatedchip surface, and observed dissociation of bound CCL20 for up to 20minutes (FIGS. 10A and B). We fitted binding data globally to a 1:1Langmuir model. The results are shown in Table 15 and represent theaverage of two independent experiments. Affinity between CCL20 and itsreceptor, CCR6, has been reported as 500 μM in primary human cells (Liaoet al., J. Immunol. 168(10):4871-4880 (2002)); these data demonstratethat the affinities of the HC2/LC3 and HC2/LK3 antibodies for CCL20 areapproximately 10 times higher than the affinity of CCR6 for CCL20.

TABLE 15 SPR results of humanized anti-CCL20 antibodies of the inventionHeavy chain/ k_(a) k_(d) K_(D) Standard Light chain (×10⁵ M⁻¹ sec⁻¹)(×10⁻⁵ sec⁻¹) (pM) deviation HC2/LC3 145 61 44 15 HC2/LK3 166 117 70 7

Example 6 Cross-Reactivity of Humanized Anti-Human CCL20 MAbs toChemokine Paralogs

We examined HC2/LK3 and HC2/LC3 for their specificity for human CCL20 byanalyzing their cross-reactivity against a chemokine panel (Table 16)using an enzyme-linked immunosorbent assay (ELISA).

TABLE 16 Recombinant human Chemokines used in ELISA assay ChemokineMaker Cat. No. CCL20 MIP-3a/LARC R&D #360-MP-025/CF XCL1 Ltn R&D#695-LT-025/CF CCL28 R&D #717-VC-025/CF CCL27 CTACK R&D #376-CT-025/CFCCL25 TECK GT #2234X CCL24 Eotaxin-2/ GT #2343X MPIF-2 CCL22 MDC R&D#336-MD-025/CF CCL21 6Ckine/SLC R&D #366-6C-025/CF CCL19 MIP-3b R&D#361-MI-025/CF CCL17 TARC GT #2364 CCL16 HCC-4 R&D #802-HC-025/CF CCL13MCP-4 GT #2327X CCL11 Eotaxin R&D #320-EO-020/CF CCL7 MARC R&D#282-P3-010/CF CCL5 Rantes CHEMICON #GF020 CCL4 MIP-1β R&D#271-BME-010/CF CCL3 MIP-1a GT #2270X CCL2 MCP-1/MCAF/JE PEPRO #300-04TECH CCL1 TCA3/I-309 GT #2272 CXCL16 CXCL16 GT #2976X CXCL13 BCA-1/BLCR&D #801-CX-025/CF CXCL12 SDF-1/PBSF R&D #350-NS-010/CF CXCL10 IP-10PEPRO #300-12 TECH CXCL9 MIG GT #2392X CXCL8 IL-8 PEPRO #200-08M TECHCXCL4 PF4 R&D #795-P4-025/CF CXCL2 GROβ R&D #276-GB-010/CF CXCL1 GROαR&D #275-GR-010/CF CX3CL1 FKN R&D #362-CX-025/CF (Chemokine Domain)CX3CL1 FKN R&D #365-FR-025/CF (Extracellular Domain)The wells of a 96-well plate were coated with 1 μg/ml of the recombinanthuman chemokines in PBS (−). After overnight incubation at 4° C., thewells were blocked with 1×Block-Ace (Dainippon Sumitomo Pharma, UK-B80)for 1 hour at room temperature. After washing twice with 0.02% Tween20/PBS (−), we added 50 μl of 10 μg/ml purified 36F7C10, chimeric36F710, HC2/LK3, or HC2/LC3 in 0.02% Tween 20/PBS (−) to each well. Thewells were incubated for 1 hour at room temperature and washed threetimes as described in Example 3. Horseradish peroxidase (HRP)-conjugatedanti-mouse IgG antibody (Jackson, #715-035-150, for 36F7C10) orHRP-conjugated anti-human IgG Fcγ fragment (Jackson, #109-035-098, forchimeric and humanized mAbs) was then added (in a 5000 fold dilutionwith 0.02% Tween 20/PBS (−)) and the wells were incubated for 1 hour atroom temperature. After washing five times, a TMBZ(3,3′,5,5′-tetramethylbenzidine) solution (1%, in N,N-Dimethylformamide)was added to the wells and incubated for 15-30 minutes. The reaction wasstopped by adding an equal volume of a 2 M H₂SO₄ stopping solution, andthe optical density was read at 450 nm by ARVO (PerkinElmer). Asdemonstrated in FIG. 11B, HC2/LK3 and HC2/LC3 bind specifically to humanCCL20 over the other chemokines in the panel.

In these assays, although HC2/LK3 was reactive against plate-bound humanCCL20, it appeared less potent than the mouse 36F7C10 or chimeric36F7C10-hFc antibodies (FIG. 11A). However, the HC2/LK3 showed clear andstrong binding against CCL20 anchored via a His-tag in ELISA assays(FIG. 11C), which allowed exposure of all CCL20 portions to the solvent.This may indicate that the CCL20 epitope(s) for HC2/LK3 are buried oroccluded in the procedure used to bind CCL20 to the plate surface in thefirst assay format. To avoid this possible artifact, we employedBiacore™ analysis in which free CCL20 was applied over MAbs captured ona sensor chip. In the Biacore™ assays, HC2/LK3 and HC2/LC3 showed strongCCL20 binding comparable to that of the 36F7C10-hFc chimeric antibody(FIG. 11D). Further, the small reaction against CXCL4 observed in FIG.11B was found to be negligible in the Biacore™ assays (FIG. 11D).

Phylogenetic analysis by Dereeper et al., (Nucleic Acids Res.1(36):W465-469 (2008)) indicates that CCL16 is the chemokine closest insequence to CCL20, although the percent identity between CCL20 and CCL16is less than 37.5% (homology in only 56 out of 70 amino acids comprisingthe mature CCL20 peptide (SIM—Alignment Tool for protein sequences,Swiss Institute of Bioinformatics)) (FIG. 12). We used Biacore™ analysisto test whether HC2/LK3 and HC2/LC3 cross-react with CCL16. Monoclonalmouse anti-human Fc was immobilized on all four flow cells of a CM5 chipat a flow rate of 25 μl/min with HBS-EP buffer containing 0.2 mg/mL BSA.Subsequently, 50 μl of a 1 μg/ml solution of chimeric, HC2/LC3, orHC2/LK3 antibody in HBS-EP buffer with 0.2 mg/ml BSA was injected overflow cells 2, 3, and 4, respectively. 150 μl of a 100 nM solution ofCCL20 or CCL16 in HBS-EP buffer (or buffer alone) with 0.2 mg/ml BSA wasthen injected over all four flow cells at a flow rate of 40 μl/min.Dissociation followed for 20 min. Flow cell 1 (anti-human Fc antibodyalone) was used as a reference for all flow cells.

Under these conditions, human CCL20 bound to the chimeric (FIG. 13A),HC2/LC3 (FIG. 13B), and HC2/LK3 (FIG. 13B) antibodies captured on thechip with similar strength. By contrast, no significant binding wasdetected when human CCL16 was run through the chip, indicating thatalthough CCL16 is the chemokine closest in sequence to CCL20, CCL20 andCCL16 do not possess sufficient homology (<37%, as discussed above) toallow the humanized anti-CCL20 antibodies to cross-react with CCL16.

These data indicate that HC2/LK3 and HC2/LC3 are specific for humanCCL20 over other chemokines.

Example 7 Cross-Reactivity of Humanized Anti-Human CCL20 MAbs to SpeciesOrthologs

We then determined the cross-reactivity of the HC2/LC3 and HC2/LK3 MAbsto CCL20 from other species. Amino acid sequence alignments among CCL20orthologs (obtained using SIM—alignment tool for protein sequences(Swiss Institute of Bioinformatics)) indicate that the identity betweencynomolgus/rhesus and human CCL20 is 86%, while the homology betweenmouse and human CCL20 is 64% (FIG. 14; human CCL20—SEQ ID NO: 85; rhesusCCL20—SEQ ID NO: 86; cynomolgus CCL20—SEQ ID NO: 87; partial mouseCCL20—SEQ ID NO: 88 (the entire mouse CCL20 amino acid sequence may befound in SEQ ID NO: 102; residues 1-27 constitute the signal sequence;see Table 18)).

To test whether the chimeric and humanized antibodies can bind to mouse,cynomolgus, or rhesus macaque CCL20 orthologs, we used ELISA assays asillustrated in FIG. 15. Recombinant secreted alkaline phosphatase(SEAP)-chemokine fusion proteins were engineered as described in Example2, expressed and purified for all of the CCL20 orthologs tested. NuncMaxiSorb flat-bottom black plates were coated with 1 μg/ml mouseanti-placental alkaline phosphatase antibody (Pierce, cat #MA1-19354) in50 mM sodium bicarbonate, pH 9.4, overnight at 4° C. Plates were thenblocked using 1× phosphate buffered saline with Tween-20 (PBST) with 5%bovine serum albumin (BSA) for 2 hours at room temperature. 20 nMSEAP-CCL20 fusion proteins were serially diluted 5-fold in Opti-MEMmedium (Invitrogen). Chimeric and humanized (HC2/LC3 and HC2/LK3)antibodies were diluted to 1 μg/ml in 1×PBST with 5% BSA, added to theplates, and incubated for 1 hr at room temperature. The plates were thenwashed with 1×PBST with 5% BSA three times. Goat anti-human IgG+IgM(H+L) HRP conjugate (Jackson ImmunoResearch cat #109-035-127) wasdiluted to 80 ng/ml, added to the plate, and incubated for 1 hour atroom temperature. QuantaBlu fluorescent HRP substrate (Pierce cat#15169) was added for detection of bound antibody by measuringfluorescence in a Molecular Devices M5 (Excitation 325 nm/Emission 420nm) plate reader.

While the hamster anti-mouse CCL20 antibody 2F5-5 MAb bound mouse CCL20with a 50% effective dose (EC₅₀) of 64 μM (data not shown), neither thechimeric nor humanized anti-human CCL20 antibodies bound detectably tomouse CCL20 or rat CCL20 under the conditions described above. Bycontrast, both the chimeric and humanized antibodies effectively boundto human, rhesus, and cynomolgus CCL20 (FIGS. 16A-C, 17A and B). Whilethe EC₅₀ for human and rhesus CCL20 appeared similar (for example, 62and 101 pM for HC2/LC3), the EC₅₀ for cynomolgus CCL20 was 340 pM forHC2/LC3, a 5.4 fold difference versus human CCL20. Similar results wereseen for HC2/LK3. This suggests that a much greater concentration ofantibody was required to achieve the same amount of binding tocynomolgus CCL20 as to human or rhesus CCL20.

Example 8 Surface Plasmon Resonance Assays for Cross-Reactivity ofHumanized Anti-CCL20 MAbs To CCL20 Species Orthologs

We also used surface plasmon resonance (Biacore™) analysis to testwhether HC2/LC3 and HC2/LK3 can bind to mouse, cynomolgus, or rhesusmacaque CCL20 orthologs. Monoclonal mouse anti-human placental alkalinephosphatase was immobilized on all four flow cells of a CM5 chip at aflow rate of 25 μl/min. 25 μl of a 2 nM solution of human, rhesus,cynomolgus, or mouse CCL20-SEAP in HBS-EP buffer was injected over flowcell 2, 3, or 4. For antibody binding, 240 μl of 0-80 nM dilutions ofHC2/LC3, HC2/LK3, or 2F5-5 MAb in HBS-EP buffer (or buffer alone) wasinjected over all four flow cells at a flow rate of 50 μl/min.Dissociation followed for 45 min. Flow cell 1 (anti-SEAP alone) was usedas a reference for all flow cells. Regeneration of the flow cells wasperformed with 10 mM glycine at pH 2.25. Due to regeneration effects onthe capturing capacity of the chips, antibody injections were performedsequentially from low to high concentration. Data fitting was performedusing a 1:1 Langmuir model.

Under the above conditions, hamster anti-mouse CCL20 antibody 2F5-5bound mouse CCL20 with a (bivalent) K_(D) of 4.9 μM, but did notsignificantly bind human, rhesus, or cynomolgus CCL20. In contrast,chimeric, HC2/LC3, and HC2/LK3 antibodies effectively bound to human,rhesus, and cynomolgus CCL20 (Table 17).

The apparent affinity values measured were higher in this assay thanpreviously detected in Example 5 (for example 4.7 pM for HC2/LC3 in thepresent assay vs. 44 pM in Table 3) due to the bivalent (higheraffinity) nature of the assay format used (versus the monovalent formatused for the data shown in Table 15). The K_(D) values for rhesus andcynomolgus CCL20 were generally higher (3-fold) than for human CCL20,indicating that the antibodies bound more specifically to human CCL20.In contrast to the ELISA data from Example 7, however, we observed nosignificant difference between binding affinity for rhesus andcynomolgus CCL20.

TABLE 17 Assessment of anti-CCL20 humanized monoclonal antibody bindingto CCL20 orthologs by Biacore ™ k_(a) k_(d) K_(D) Antibody Ligand (×10⁵M⁻¹sec⁻¹) (×10⁻⁵ sec⁻¹) (pM) Chimeric hCCL20 29 2.08 7.16 clone rCCL2029.4 1.78 6.05 36F7C10^(a) cCCL20 38.3 5.04 13.2 HC2/LC3 hCCL20 56.92.68 4.71 rCCL20 41.3 7.51 18.2 cCCL20 86.4 13.4 15.5 HC2/LK3 hCCL2028.8 3.39 11.8 rCCL20 26.3 8.21 31.2 cCCL20 52.8 14.9 28.3 HamsterhCCL20 No binding No binding No binding anti-mouse rCCL20 No binding Nobinding No binding 2F5-5^(b) cCCL20 No binding No binding No bindingmCCL20 9.63 0.474 4.92 ^(a)Previously shown not to bind mCCL20^(b)Hamster anti-mouse data from earlier chip

Example 9 Epitope Mapping for Mouse Anti-Human CCL20 Clone 36F7C10

To determine the human CCL20 epitope(s) to which mouse anti-human CCL20monoclonal antibody 36F7C10 binds, we used a hydrogen/deuterium exchangemethod in which antibody binding protects and thus preserves deuterationof the epitope. As shown in FIG. 18A, we observed very strongperturbations near the N-terminus of human CCL20 at residues 7-9, 10-19,and 20-22, possibly representing the epitope(s). Marginal protection wasalso observed near the C-terminus at residues 39-55, 56-67, and 61-70.These segments may be peripheral to the epitope, or their breathingmotions may be conjugated to the epitope. The deuteration level of eachindicated amino acid is shown at four timepoints: from top to bottom,150, 500, 1,500, and 5,000 s.

The regions identified as the epitope for 36F7C10 fall within theN-terminal and loop regions of CCL20, which are known to be critical forCCR6 binding and signaling (Malik et al., Acta Cryst F62:631-634(2006))(FIG. 18B). Chimeric and humanized antibodies derived from36F7C10, especially antibodies with the same heavy and light chain CDR1,CDR2 and CDR3 amino acid sequences as 36F7C10, or similar heavy andlight chain CDR1, CDR2 and CDR3 amino acid sequences as 36F7C10 (e.g.,with less than 3, 2 or 1 amino acid substitutions as compared to theCDRs of 36F7C10) are expected to bind to the same epitope. Theidentification of this epitope may thus explain the ability of the36F7C10 MAb and chimeric and humanized antibodies derived therefrom toneutralize CCL20 activity.

Example 10 In Vivo Chemotaxis Assay for Humanized Anti-Human CCL20Antibodies

To test whether HC2/LC3 and HC2/LK3 can inhibit CCL20-induced chemotaxisin vivo, we took advantage of the fact that human CCL20 can interactwith mouse CCR6 (SEQ ID NO: 106) to induce chemotaxis of mouse T cells(FIG. 19). We used a hybrid in vivo system measuring the migration ofmouse T cells towards intradermally injected human CCL20 (FIG. 20).

Recombinant human CCL20 (10 ng/head) and vehicle were intradermallyinjected into the shaved skin on the right and left sides, respectively,of the backs of the test mice (groups of n=3). Calcein-AM-labeled mousesplenic T cells (5×10⁶ cells/mouse) were then transferred intravenouslyinto the tail vein, with simultaneous injection of the indicatedantibodies into the tail vein at the dosages described in FIG. 20. Afterone hour, the fluorescent positive cells were counted using astereoscopic microscope at the site of the intradermal injections.

Both HC2/LK3 and HC2/LC3 significantly inhibited cell migration to theCCL20-injected sites at a level comparable to that of parental mouseantibody 36F7C10 and its chimeric form. Because the chemotaxis of Tcells is a key step in the inflammatory cascade, prevention of thismigration has clinical implications for the treatment of autoimmune andinflammatory conditions.

Example 11 Effect of 2F5-5 MAb on Type II Collagen-Induced Arthritis

To further evaluate the use of anti-CCL20 antibodies for therapeuticindications, we performed further in vivo studies in mice using thehamster anti-mouse 2F5-5 MAb. First, we evaluated the ability of 2F5-5to neutralize CCL20-mediated chemotaxis in CIA mice (an animal model ofrheumatoid arthritis). CIA was induced essentially as described inExample 1. After the development of arthritis (arthritis score 1-3),mice were randomized and treated with 500 μg/mouse of either 2F5-5 MAbor a control IgG antibody. In both cases, the antibody was administeredintravenously every other day. Compared to the control IgG, 2F5-5 MAbinhibited the further development of arthritis symptoms (FIGS. 21, 22).

We also used X-ray scoring to determine the effects of 2F5-5 MAb on bonelesions, which are frequently seen in rheumatoid arthritis. Scoring wasperformed as described in Inoue et al., Agents Actions 39:187-194(1993). Briefly, each paw of a CIA mouse was graded on a scale of 0 to 3based on the severity of osteoporosis (O), bone erosion (E), and newbone formation (N) according to X-ray images. The scale used was: 0, nochange; 1, slight change; 2, moderate change; and 3, severe change. Thescores for each factor were added to develop a cumulative X-ray scorefor bone lesions. Mice treated with 2F5-5 MAb demonstrated remarkablyless severe X-ray scores on day 39, as compared to mice treated with acontrol IgG (FIGS. 23, 24).

We confirmed the effect of 2F5-5 MAb treatment on bone pathology byanalyzing several biomarkers using ELISA. CIA mice were immunized andtreated with 500 μg of 2F5-5 or control IgG antibody as described above.Plasma samples were prepared from the mice on day 11 or 12 after thesecond immunization. A marker of cartilage destruction, serum cartilageoligomeric matrix protein (COMP), was quantified using the Animal COMPELISA Enzyme immunoassay kit (AnaMar Medical) according to themanufacturer's instructions (incorporated herein by reference), exceptthat animal plasma samples were diluted 1:20. Because COMP is pre-coatedon the plate, this is a competition ELISA, whereby addition of the COMPstandard or COMP-containing plasma results in a decrease in OD₄₅₀ (FIG.25A). Raw data from a sample assay is shown in FIGS. 25B and 25C. COMPserum levels in CIA mice were reduced almost to normal levels bytreatment with 2F5-5 MAb (FIG. 26), demonstrating the ability of 2F5-5to inhibit cartilage destruction.

Because the formation and differentiation of osteoclasts is responsiblefor RA-related osteoporosis and erosion, we measured the levels ofosteoclast induction molecule receptor activator for nuclear factor κBligand (RANKL), and osteoclast markers such as the receptor activatorfor nuclear factor κB (RANK), tartrate resistant acid phosphatase(TRAP), and cathepsin K, as indicators for arthritic therapy. Weevaluated mRNA expression levels of these markers by quantitative PCRusing total RNA isolated from homogenized mouse paws. The paws werefirst homogenized with a homogenizer after soaking in a tissue lysingbuffer containing Trizol reagent (Invitrogen, CA, USA). After addingchloroform, samples were centrifuged at 14,000 rpm for 15 minutes at 4°C. to separate the solution into aqueous and organic phases. The aqueousphase was removed and isopropanol added to it, followed bycentrifugation at 14,000 rpm for 15 minutes at 4° C. to obtain RNApellets. RNA pellets dissolved with RNAse free water were used in anRNAeasy mini kit (QIAGEN, Valencia, Calif., USA) to isolate the RNA andtreated with DNAse to remove any DNA. Complimentary DNA (cDNA) wasgenerated from the RNA with an RT reaction kit (RNA PCR Kit, TAKARA Bio,Inc. Shiga, Japan) according to the manufacturer's protocol.Quantitative real time PCR for each cDNA species was performed andcompared to the level of a housekeeping gene, hypoxanthine guaninephosphoribosyl transferase (HPRT). The following forward and reverseprimer sets were used in the PCR reactions:

RANKL, (SEQ ID NO: 89) 5′-CATTTGCACACCTCACCATC-3′ and (SEQ ID NO: 90)5′-TCCGTTGCTTAACGTCATGT-3′; RANK, (SEQ ID NO: 91)5′-CGGCGTTTACTACAGGAAGG-3′ and (SEQ ID NO: 92)5′-TTCTTGCTGACTGGAGGTTG-3′; TRAP, (SEQ ID NO: 93)5′-GCTGGAAACCATGATCACCT-3′ and (SEQ ID NO: 94)5′-GGTAGTAAGGGCTGGGGAAG-3′; Cathepsin K, (SEQ ID NO: 95)5′-CAGTGTTGGTGGTGGGCTAT-3′ and (SEQ ID NO: 96)5′-CCGAGCCAAGAGAGCATATC-3′; and HPRT, (SEQ ID NO: 97)5′-CAGGCCAGACTTTGTTGGAT-3′ and (SEQ ID NO: 98)5′-TTGCGCTCATCTTAGGCTTT-3′.All primers were designed using web-based software Primer 3 (WhiteheadInstitute for Biomedical Research, Cambridge, Mass., USA) to avoidnon-specific amplification of RNA. Reaction mixtures with cDNA template,primers, uracil DNA glycosylase (Invitrogen, CA, USA), and QuantiTectSYBR Green PCR Master Mix (QIAGEN, Valencia, Calif., USA) were used inthe amplification reaction in an ABI PRISM 7700 Sequence DetectionSystem (Applied Biosystems, Foster, Calif., USA). Expression levels wereautomatically quantified by ABI PRISM 7700 Sequence Detector Software.

Following 2F5-5 MAb treatment, mRNA levels were suppressed for allmarkers in the joint tissue (FIGS. 27A and B; show fold change inexpression level compared to housekeeping gene HPRT), providing furtherevidence that 2F5-5 inhibits bone pathology in vivo.

Example 12 Effects of 2F5-5 MAb on Glucose-6-Phosphate Isomerase-InducedArthritis

We tested the anti-arthritic effects of 2F5-5 MAb treatment in mice withglucose-6-phosphate isomerase (G6PI)-induced arthritis, another mousemodel for RA. Arthritis was induced by intradermal sensitization withrecombinant GST-G6PI (300 μg/mouse) emulsified in complete Freund'sadjuvant and injected at the base of the tail of DBA/1 mice. Six daysafter the G6PI immunization and just prior to the onset of jointswelling, mice were randomized and treated with 500 μg/mouse of eitherisotype-matched antibody or 2F5-5 MAb. The severity of arthriticsymptoms in the paws of each mouse was graded as described previously inExample 1. Mice treated with 2F5-5 MAb exhibited significantly lowerarthritic scores than mice treated with isotype-matched antibodies,showing that 2F5-5 strongly suppresses arthritis development compared tothe control (FIG. 28).

These data demonstrate the efficacy of anti-CCL20 antibody treatment inin vivo arthritis models, and suggest that the use of anti-CCL20antibodies may be beneficial in the treatment of rheumatoid arthritis.

Example 13 Effect of 2F5-5 MAb on Oxazolone-Induced Atopic Dermatitis

We then evaluated the effect of 2F5-5 MAb treatment in mouse models ofdermatitis. In one model, oxazolone was used to induce atopic dermatitisin mice prone to the disease (NC/Nga strain). The abdominal skin of themice was shaved and exposed to oxazolone on days 0, 5, and 8. On day 13,mice showing signs of dermatitis (score 2) were selected and immunizedagain with oxazolone. Mice were thereafter randomized in groups of sixfor treatment with either 2F5-5 MAb or an isotype-matched control IgGantibody (500 μg/mouse, administered intravenously every other day). Weassayed dermatitis scores in a blind study as follows: each dermatitissymptom such as dryness, scale, erythema, oozing/crusting, andexcoriation was scored on a scale of 0 to 3 (0=none, 1=slight,2=moderate, 3=severe); these scores were then added for a cumulativedermatitis score (as described in Leung et al., J. Allergy Clin.Immunol. 85(5):927-933 (1990)). We compared the magnitude of diseasesuppression between the two groups by quantifying the area under thecurve (“AUC”) from day 13 to day 18. In this dermatitis model, 2F5-5 MAbinduced a statistically significant (p<0.05) suppression of diseaseprogression compared to control IgG (FIG. 29).

Example 14 Effect of 2F5-5 MAb on DNFB-Induced Allergic ContactDermatitis

We tested the effect of 2F5-5 MAb treatment in dinitrofluorobenzene(DNFB)-induced allergic contact dermatitis, a second mouse model ofdermatitis. Mice were divided into groups of 6 and sensitized bybrushing 25 μl of DNFB solution (0.4% DNFB in a 4:1 solution ofacetone:olive oil) on the shaved abdomen for two successive days (days 0and 1). One group was treated with 2F5-5 MAb, while the other group wastreated with an isotype-matched control antibody (500 μg/mouseintravenously on days 0, 2, and 5). On day 5, the mice werere-challenged by applying 20 μl of 0.2% DNFB solution (in 4:1acetone:olive oil) to one side of one ear. Ear thickness was measured asan indicator of edema on day 6 using a thickness gauge. Treatment with2F5-5 MAb reduced ear thickness, indicating successful prevention ofdermatitis development (FIG. 30).

All publications, patents, and patent applications cited in thisspecification are incorporated herein by reference. Although theforegoing invention has been described in some detail by way ofillustration and example for purposes of clarity of understanding, itwill be readily apparent to those of ordinary skill in the art in lightof the teachings of this invention that certain changes andmodifications may be made thereto without departing from the spirit orscope of the appended claims.

TABLE 18 Amino acid and nucleotide sequences for human and mouse CCL20and CCR6 Sequence Sequence SEQ ID Description Type (signal sequencesunderlined) NO: Human CCL20 Amino Acidmcctkslllaalmsvlllhlcgeseaasnfdcclgytdri 85 with signallhpkfivgftrqlanegcdinaiifhtkkklsvcanpkqt sequence wvkyivrllskkvknmNucleotide atgtgctgtaccaagagtttgctcctggctgctttgatgt 101cagtgctgctactccacctctgcggcgaatcagaagcagcaagcaactttgactgctgtcttggatacacagaccgtattcttcatcctaaatttattgtgggcttcacacggcagctggccaatgaaggctgtgacatcaatgctatcatctttcacacaaagaaaaagttgtctgtgtgcgcaaatccaaaacagacttgggtgaaatatattgtgcgtctcctcagtaaaaaagtca agaacatg Human CCL20 Amino Acidasnfdcclgytdrilhpkfivgftrqlanegcdinaiifh 99 without signaltkkklsvcanpkqtwvkyivrllskkvknm sequence Nucleotidegcaagcaactttgactgctgtcttggatacacagaccgta 100ttcttcatcctaaatttattgtgggcttcacacggcagctggccaatgaaggctgtgacatcaatgctatcatctttcacacaaagaaaaagttgtctgtgtgcgcaaatccaaaacagacttgggtgaaatatattgtgcgtctcctcagtaaaaaagt caagaacatg Mouse CCL20 AminoAcid macggkrllflalawvllahlcsqaeaasnydcclsyiqt 102 with signalplpsraivgftrqmadeacdinaiifhtkkrksvcadpkq sequence nwvkravnllslrvkkmNucleotide atggcctgcggtggcaagcgtctgctcttccttgctttgg 103catgggtactgctggctcacctctgcagccaggcagaagcagcaagcaactacgactgttgcctctcgtacatacagacgcctcttccttccagagctattgtgggtttcacaagacagatggccgatgaagcttgtgacattaatgctatcatctttcacacgaagaaaagaaaatctgtgtgcgctgatccaaagcagaactgggtgaaaagggctgtgaacctcctcagcctaagag tcaagaagatg Human CCR6 AminoAcid msgesmnfsdvfdssedyfvsvntsyysvdsemllcslqe 104vrqfsrlfvpiayslicvfgllgnilvvitfafykkarsmtdvyllnmaiadilfvltlpfwayshatgawvfsnatckllkgiyainfncgmllltcismdryiaivqatksfrlrsrtlprskiiclvvwglsviissstfvfnqkyntqgsdvcepkyqtvsepirwkllmlglellfgffiplmfmifcytfivktlvqaqnskrhkairviiavvlvflacqiphnmvllvtaanlgkmnrscqsekligytktvtevlaflhcclnpvlyafigqkfrnyflkilkdlwcvrrkykssgfscagrysenisrqt setadndnassftm Nucleotideatgagcggggaatcaatgaatttcagcgatgttttcgact 105ccagtgaagattattttgtgtcagtcaatacttcatattactcagttgattctgagatgttactgtgctccttgcaggaggtcaggcagttctccaggctatttgtaccgattgcctactccttgatctgtgtctttggcctcctggggaatattctggtggtgatcacctttgctttttataagaaggccaggtctatgacagacgtctatctcttgaacatggccattgcagacatcctctttgttcttactctcccattctgggcagtgagtcatgccaccggtgcgtgggttttcagcaatgccacgtgcaagttgctaaaaggcatctatgccatcaactttaactgcgggatgctgctcctgacttgcattagcatggaccggtacatcgccattgtacaggcgactaagtcattccggctccgatccagaacactaccgcgcagcaaaatcatctgccttgttgtgtgggggctgtcagtcatcatctccagctcaacttttgtcttcaaccaaaaatacaacacccaaggcagcgatgtctgtgaacccaagtaccagactgtctcggagcccatcaggtggaagctgctgatgttggggcttgagctactctttggtttctttatccctttgatgttcatgatattttgttacacgttcattgtcaaaaccttggtgcaagctcagaattctaaaaggcacaaagccatccgtgtaatcatagctgtggtgcttgtgtttctggcttgtcagattcctcataacatggtcctgcttgtgacggctgcaaatttgggtaaaatgaaccgatcctgccagagcgaaaagctaattggctatacgaaaactgtcacagaagtcctggctttcctgcactgctgcctgaaccctgtgctctacgcttttattgggcagaagttcagaaactactttctgaagatcttgaaggacctgtggtgtgtgagaaggaagtacaagtcctcaggcttctcctgtgccgggaggtactcagaaaacatttctcggcagaccagtgagaccgcagataacgacaatgcgtcgtccttcacta tg Mouse CCR6 Amino Acidmnstesyfgtddydnteyysippdhgpcsleevrnftkvf 106vpiayslicvfgllgnimvvmtfafykkarsmtdvyllnmaitdilfvltlpfwavthatntwvfsdalcklmkgtyavnfncgmlllacismdryiaivqatksfrvrsrtlthskvicvavwfisiiissptfifnkkyelqdrdvcepryrsysepitwkllgmglelffgfftpllfmvfcylfiiktivqaqnskrhrairvviavvlvflacqiphnmvllvtavntgkvgrscstekvlaytrnvaevlaflhcclnpvlyafigqkfrnyfmkimkdvwcmrrknkmpgflcarvysesyisrqtsetvend nassftm Nucleotideatgaattccacagagtcctactttggaacggatgattatg 107acaacacagagtattattctattcctccagaccatgggccatgctccctagaagaggtcagaaacttcaccaaggtatttgtgccaattgcctactccttaatatgtgtctttggcctcctgggcaacattatggtggtgatgacctttgccttctacaagaaagccagatccatgactgacgtctacctgttgaacatggccatcacagacatactctttgtcctcaccctaccgttctgggcagttactcatgccaccaacacttgggttttcagcgatgcactgtgtaaactgatgaaaggcacatatgcggtcaactttaactgtgggatgctgctcctggcctgtatcagcatggaccggtacattgccatcgtccaggcaaccaaatctttccgggtacgctccagaacactgacgcacagtaaggtcatctgtgtggcagtgtggttcatctccatcatcatctcaagccctacatttatcttcaacaagaaatacgagctgcaggatcgtgatgtctgtgagccacggtacaggtctgtctcagagcccatcacgtggaagctgctgggtatgggactggagctgttctttgggttcttcacccctttgctgtttatggtgttctgctatctgttcattatcaagaccttggtgcaggcccagaactccaagaggcacagagcaatccgagtcgtgatcgctgtggttctcgtgttcctggcttgtcagatccctcacaacatggtcctcctcgtgactgcggtcaacacgggcaaagtgggccggagctgcagcaccgagaaagtcctcgcctacaccaggaacgtggccgaggtcctggctttcctgcattgctgcctcaaccccgtgttgtatgcgtttattggacagaaattcagaaactacttcatgaagatcatgaaggatgtgtggtgtatgagaaggaagaataagatgcctggcttcctctgtgcccgggtttactcggaaagctacatctccaggcagaccagtgagaccgtcgaaaatgat aatgcatcgtcctttaccatg

TABLE 19 Sequence Identification Numbers SEQ ID NO Description 1 HC2Full-Length Heavy Chain Amino Acid Sequence 2 HC3 (HC) 3 36HKK3 4 42HKK15 42HKK2 6 42HKK3 7 LC3 Full-Length Light Chain 8 LK3 (LC) 9 HC2 HeavyChain Variable Domain 10 HC3 (VH) 11 36HKK3 12 42HKK1 13 42HKK2 1442HKK3 15 LC3 Light Chain Variable Domain 16 LK3 (VL) 17 HC2 Full-LengthHeavy Chain Nucleotide Sequence 18 HC3 (HC) 19 36HKK3 20 42HKK1 2142HKK2 22 42HKK3 23 LC3 Full-Length Light Chain 24 LK3 (LC) 25 HC2 HeavyChain Variable Domain 26 HC3 (VH) 27 36HKK3 28 42HKK1 29 42HKK2 3042HKK3 31 LC3 Light Chain Variable Domain 32 LK3 (VL) 33 36F7C10 HeavyChain Signal Sequence Amino Acid Sequence 34 Light Chain Signal Sequence35 40-1C10B9 Heavy Chain Signal Sequence 36 Light Chain Signal Sequence37 42G5B10 Heavy Chain Signal Sequence 38 Light Chain Signal Sequence 3936F7C10 VH 40 VL 41 40-1C10B9 VH 42 VL 43 42G5B10 VH 44 VL 45 36F7C10Heavy Chain Signal Sequence Nucleotide Sequence 46 Light Chain SignalSequence 47 40-1C10B9 Heavy Chain Signal Sequence 48 Light Chain SignalSequence 49 42G5B10 Heavy Chain Signal Sequence 50 Light Chain SignalSequence 51 36F7C10 VH 52 VL 53 40-1C10B9 VH 54 VL 55 42G5B10 VH 56 VL57 IGHV1-46*03 Germline Sequence 58 JH4 Germline Sequence 59IGKV1D-39*01 Germline Sequence 60 Mouse 36F7C10 H-CDR1 NYWMH HC2 (Kabat)HC3 36HKK3 61 Mouse 42G5B10 SYWMH 42HKK1 42HKK2 42HKK3 62 IGHV1-46*03/SYYMH ABM67212 63 Mouse 36F7C10 H-CDR2 VIDPSDSYTTYNQKFKG HC3 (Kabat)36HKK3 64 HC2 VIDPSDSYTTYAQKFQG 65 Mouse 42G5B10 LIDPSDKYTNYNQKFKG42HKK1 42HKK2 42HKK3 66 IGHV1-46*03/ IINPSGGSTSYAQKFQG ABM67212 67 Mouse36F7C10 H-CDR3 GNYGVDYAMDY HC2 (Kabat or Chothia) HC3 36HKK3 68 Mouse42G5B10 GNYGVDYGMDY 42HKK1 42HKK2 42HKK3 69 ABM67212 EGDGYIQAFDY 70Mouse 36F7C10 L-CDR1 GASENIYGALN LK3 (Kabat or Chothia) 71 LC3RASENIYGALN 72 IGKV1D-39*01/ RASQSISSYLN BAH04867.1 73 Mouse 36F7C10L-CDR2 GATNLAD LC3 (Kabat or Chothia) LK3 74 IGKV1D-39*01/ AASSLQSBAH04867.1 75 Mouse 36F7C10 L-CDR3 QNVLITPYT LC3 (Kabat or Chothia) LK376 BAH04867.1 QQSYSTPYT 77 Mouse 36F7C10 H-CDR1 GYTFTNY HC2 (Chothia)HC3 36HKK3 78 Mouse 42G5B10 GYTFTSY 42HKK1 42HKK2 42HKK3 IGHV1-46*03/ABM67212 79 Mouse 36F7C10 H-CDR2 VIDPSDSYTT HC2 (Chothia) HC3 36HKK3 80Mouse 42G5B10 LIDPSDKYTN 42HKK1 42HKK2 42HKK3 81 IGHV1-46*03/ IINPSGGSTSABM67212 82 ABM67212 VH 83 BAH04867.1 VL 84 Human CCL20 variant (withoutsignal sequence) amino acid sequence 85 Human CCL20 (with signalsequence) amino acid sequence 86 Rhesus CCL20 amino acid sequence 87Cynomolgus CCL20 amino acid sequence 88 Mouse CCL20 amino acid sequence(partial) 89 5′-CATTTGCACACCTCACCATC-3′ RANKL Primers 905′-TCCGTTGCTTAACGTCATGT-3′ 91 5′-CGGCGTTTACTACAGGAAGG-3′ RANK 925′-TTCTTGCTGACTGGAGGTTG-3′ 93 5′-GCTGGAAACCATGATCACCT-3′ TRAP 945′-GGTAGTAAGGGCTGGGGAAG-3′ 95 5′-CAGTGTTGGTGGTGGGCTAT-3′ Cathepsin K 965′-CCGAGCCAAGAGAGCATATC-3′ 97 5′-CAGGCCAGACTTTGTTGGAT-3′ HPRT 985′-TTGCGCTCATCTTAGGCTTT-3′ 99 Human CCL20 (without signal sequence)amino acid sequence 100 Human CCL20 (without signal sequence) nucleotidesequence 101 Human CCL20 (with signal sequence) nucleotide sequence 102Mouse CCL20 (with signal sequence) amino acid sequence 103 Mouse CCL20(with signal sequence) nucleotide sequence 104 Human CCR6 amino acidsequence 105 Human CCR6 nucleotide sequence 106 Mouse CCR6 amino acidsequence 107 Mouse CCR6 nucleotide sequence 108 HC2 full-length aminoacid sequence (without signal sequence) 109 HC2 full-length nucleotidesequence (without signal sequence) 110 LC3 full-length amino acidsequence (without signal sequence) 111 LC3 full-length nucleotidesequence (without signal sequence) 112 LK3 full-length amino acidsequence (without signal sequence) 113 LK3 full-length nucleotidesequence (without signal sequence) 114 partial CCL20 amino acid sequence115 partial CCL16 amino acid sequence

TABLE 20 Humanized and Mouse Antibody Sequences Amino Acid SequencesH-CDR L-CDR Nucleotide Sequences HC LC VH VL 1 2 3 1 2 3 HC LC VH VLHumanized HC2 1 9 K: 60 K: 64 K: 67 17 25 Anti-Human C: 77 C: 79 C: 67CCL20 Ab HC3 2 10 K: 60 K: 63 K: 67 18 26 Chains C: 77 C: 79 C: 6736HKK3 3 11 K: 60 K: 63 K: 67 19 27 C: 77 C: 79 C: 67 42HKK1 4 12 K: 61K: 65 K: 68 20 28 C: 78 C: 80 C: 68 42HKK2 5 13 K: 61 K: 65 K: 68 21 29C: 78 C: 80 C: 68 42HKK3 6 14 K: 61 K: 65 K: 68 22 30 C: 78 C: 80 C: 68LC3 7 15 71 73 75 23 31 LK3 8 16 70 73 75 24 32 Mouse 36F7C10 39 40 K:60 K: 63 K: 67 70 73 75 51 52 Anti-Human C: 77 C: 79 C: 67 CCL20 Abs40-1C10B9 41 42 53 54 42G5B10 43 44 K: 61 K: 65 K: 68 55 56 C: 78 C: 80C: 68

What is claimed is:
 1. An isolated nucleic acid molecule encoding a) theheavy chain of a monoclonal anti-human CCL20 antibody or anantigen-binding portion thereof, wherein the complementarity determiningregion (CDR) 1, CDR2, and CDR3 of said heavy chain comprise the aminoacid sequences shown in i) SEQ ID NOS: 60, 64, and 67, respectively, orii) SEQ ID NOS: 77, 79, and 67, respectively; and b) the light chain ofthe monoclonal anti-human CCL20 antibody or antigen-binding portion,wherein the CDR1, CDR2, and CDR3 of said light chain comprise the aminoacid sequences shown in i) SEQ ID NOS: 70, 73, and 75, respectively, orii) SEQ ID NOS: 71, 73, and 75, respectively.
 2. The isolated nucleicacid molecule of claim 1, wherein the heavy chain comprises the aminoacid sequence of SEQ ID NO: 1, 9, or 108; or said amino acid sequencewithout a signal sequence, if present; or said amino acid sequencewithout a C-terminal lysine, if present.
 3. The isolated nucleic acidmolecule of claim 2, wherein the light chain comprises the amino acidsequence of SEQ ID NO: 7, 8, 15, 16, 110, or
 112. 4. The isolatednucleic acid molecule of claim 1, wherein the light chain comprises theamino acid sequence of SEQ ID NO: 7, 8, 15, 16, 110, or
 112. 5. Anisolated nucleic acid molecule encoding the amino acid sequence of SEQID NO: 1, 9, or 108, or said amino acid sequence without the C-terminallysine, if present.
 6. An isolated nucleic acid molecule encoding theamino acid sequence of SEQ ID NO: 7, 8, 15, 16, 110, or
 112. 7. Anisolated nucleic acid molecule comprising the nucleotide sequence of SEQID NO: 17, 25, or 109, or said nucleotide sequence without the sequenceencoding the heavy chain C-terminal lysine, if present.
 8. An isolatednucleic acid molecule comprising the nucleotide sequence of SEQ ID NO:23, 24, 31, 32, 111, or
 113. 9. An isolated vector comprising theisolated nucleic acid molecule of any one of claims 1-8.
 10. An isolatedhost cell comprising the isolated vector of claim
 9. 11. An isolatedhost cell comprising a) a first nucleotide sequence encoding the heavychain of a monoclonal anti-human CCL20 antibody or an antigen-bindingportion thereof, wherein the complementarity determining region (CDR) 1,CDR2, and CDR3 of said heavy chain comprise the amino acid sequencesshown in i) SEQ ID NOS: 60, 64, and 67, respectively, or ii) SEQ ID NOS:77, 79, and 67; and b) a second nucleotide sequence encoding the lightchain of the monoclonal anti-human CCL20 antibody or antigen-bindingportion, wherein the CDR1, CDR2, and CDR3 of said light chain comprisethe amino acid sequences shown in i) SEQ ID NOS: 70, 73, and 75,respectively, or ii) SEQ ID NOS: 71, 73, and 75, respectively.
 12. Thehost cell of claim 11, wherein the first and second nucleotide sequencesare located on separate nucleic acid molecules.
 13. A method of makingan anti-human CCL20 antibody or an antigen-binding portion thereof,comprising maintaining the isolated host cell of claim 11 underconditions appropriate for expression of the antibody or portion, andrecovering the antibody or portion produced.